Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 Å, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming ∼20 Å above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.To create antibodies capable of effectively neutralizing human immunodeficiency virus type 1 (HIV-1), the adaptive humoral response is driven to exceptional lengths (reviewed in reference 8). Indeed, the response often fails, and sera from individuals infected with HIV-1 typically display limited neutralization breadth (59). After several years of infection, however, antibodies capable of neutralizing diverse viral strains develop in 15 to 25% of infected individuals (3, 16, 32, 33, 49, 53). Details of the adaptive changes that allow for effective recognition are of direct vaccine relevance, and clues from rare neutralizing antibodies have been eagerly sought.Two broadly neutralizing antibodies, PG9 and PG16, were recently identified with single cell-sequencing techniques after direct microneutralization assessment of secreted antibody from individually plated, stimulated B cells (58). These antibodies are somatically related and appear to be derived from the same recombination of heavy and light chains. They both recognize a site on HIV-1 gp120 composed of elements from the second and third variable regions (V2 and V3). Despite the vaunted diversity of the HIV-1 gp120 envelope and the even higher sequence variability in the V2 and V3 regions (26), neutralization assays indicate that the recognized epitope is conserved in 70 to 80% of circulating viral isolates (58).To investigate the molecular features of PG9 and PG16 that account for their neutralization effectiveness, we prepared antigen-binding fragments (Fabs) of each antibody and screened for crystallization. We were able to obtain a number of crystals, and those of PG16 proved suitable for structural analysis. Determination of the PG16 structure visualized several unusual features, and structure-function analysis indicated that two features, extensive affinity maturation and an exceptionally long heavy chain-third complementarity-determining region (CDR H3), were critical to its neutralization effectiveness. Barriers to eliciting these two features provide a likely explanation for the rarity of antibodies like PG9 and PG16; understanding and overcoming such barriers may form the basis for an effective HIV-1 vaccine.     

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus that causes serious liver diseases in humans (31). More than 170 million people worldwide are seropositive for HCV and at risk for developing cirrhosis and hepatocellular carcinoma (50). HCV is a small, enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (31). Its genome encodes a single polyprotein precursor of about 3,000-amino-acid residues that is cleaved co- and posttranslationally by cellular and viral proteases to yield at least 10 mature products (31). The two envelope glycoproteins, E1 and E2, are released from the polyprotein by signal peptidase cleavages. These two proteins assemble as noncovalent heterodimers, which are retained mainly in the endoplasmic reticulum (ER) (36), and they are found as large disulfide-linked oligomers on the surfaces of HCV particles (46). HCV glycoproteins are involved in the entry process, and since they are present on the surfaces of viral particles, these proteins are the targets of neutralizing antibodies (4, 21).E1 and E2 generally contain 4 and 11 N-glycosylation sites, respectively, all of which have been shown to be modified by glycans (19). Despite variability in HCV envelope glycoprotein sequences, the four glycosylation sites of E1 and nine of E2 are highly conserved, suggesting that the glycans associated with these proteins play an essential role in the HCV life cycle (22). Using retroviral particles pseudotyped with genotype 1a (H strain) HCV envelope glycoproteins (HCVpp), recent studies have determined the potential roles played by these glycans in protein folding, HCV entry, and protection against neutralization (14, 19, 22). Indeed, the lack of glycan E1N1, E1N4, E2N8, or E2N10 strongly affects the incorporation of HCV glycoproteins into HCVpp, suggesting that these glycans are important for correct protein folding (19). Furthermore, mutation of glycosylation sites E2N2 or E2N4 alters HCVpp infectivity despite normal incorporation into pseudotyped particles, suggesting a role for the corresponding glycans in viral entry, at least in this model system (19). Finally, glycans at positions E2N1, E2N6, and E2N11 were shown to reduce the sensitivity of HCVpp to antibody neutralization as well as access of the CD81 coreceptor to its binding site on E2, suggesting that glycans also contribute to HCV evasion of the humoral immune response (14, 22).It has recently been proposed that targeting glycans could be a promising approach to inhibiting viral infection (1). Indeed, HCV, as well as several other viruses with highly glycosylated envelope proteins, can be inhibited by carbohydrate binding agents such as cyanovirin-N and pradimicin A (1, 7, 23). Furthermore, resistance against drugs that target glycans is likely to develop and will probably result in mutations at some glycosylation sites (3, 52). However, since glycans associated with viral envelope proteins play an important role in the viral life cycle, adaptation of viruses to the selective pressure of carbohydrate-binding agents will most likely come at a replicative cost to the virus (2).Although the role of HCV glycans has been studied using mutant recombinant HCV envelope glycoproteins incorporated into HCVpp, these particles do not recapitulate all the functions of HCV envelope proteins. Cell culture-derived virus (HCVcc) (32, 49, 55) assembles in an ER-derived compartment in association with very low density lipoproteins (17, 26), whereas HCVpp are assembled in a post-Golgi compartment and are not associated with lipoproteins (44). Importantly, this leads to differences between HCVpp and HCVcc in the oligomerization of the envelope glycoproteins (46). It is also important to note that the carbohydrate composition of viral glycoproteins can differ when the same virus is grown in different cell lines (13). Thus, HCVpp that are produced in 293T cells are not the most appropriate model for glycosylation studies, since HCV tropism is restricted to the liver. Furthermore, differences in envelope protein glycosylation have been observed between HCVpp and HCVcc particles (46). Differences in some HCV envelope protein functions were also observed when the HCVpp and HCVcc systems were compared (28, 29, 42, 43). The development of the HCVcc system provides, therefore, the opportunity to characterize the role of E1/E2-associated glycans in the context of authentic infectious virions. Here, we analyzed the role of E1/E2 glycans by introducing point mutations at N-linked glycosylation sites in the context of the HCVcc system. The effects of these mutations on virus replication, particle secretion, infectivity, and sensitivity to neutralizing antibodies were investigated. Our results demonstrate that several glycans play an important role in HCVcc assembly and/or infectivity and reduce access of neutralizing antibodies to their epitopes.     

HIV-1 gp120 Determinants Proximal to the CD4 Binding Site Shift Protective Glycans That Are Targeted by Monoclonal Antibody 2G12

HIV-1 R5 envelopes vary considerably in their capacities to exploit low CD4 levels on macrophages for infection and in their sensitivities to the CD4 binding site (CD4bs) monoclonal antibody (MAb) b12 and the glycan-specific MAb 2G12. Here, we show that nonglycan determinants flanking the CD4 binding loop, which affect exposure of the CD4bs, also modulate 2G12 neutralization. Our data indicate that such residues act via a mechanism that involves shifts in the orientation of proximal glycans, thus modulating the sensitivity of 2G12 neutralization and affecting the overall presentation and structure of the glycan shield.The trimeric envelope (Env) spikes on HIV-1 virions are comprised of gp120 and gp41 heterodimers. gp120 is coated extensively with glycans (9, 11, 15) that are believed to protect the envelope from neutralizing antibodies. The extents and locations of glycosylation are variable and evolving (15). Thus, while some glycans are conserved, others appear or disappear in a host over the course of infection. Such changes may result in exposure or protection of functional envelope sites and can result from selection by different environmental pressures in vivo, including neutralizing antibodies.We previously reported that HIV-1 R5 envelopes varied considerably in tropism and neutralization sensitivity (3, 4, 12-14). We showed that highly macrophage-tropic R5 envelopes were more frequently detected in brain than in semen, blood, and lymph node (LN) samples (12, 14). The capacity of R5 envelopes to infect macrophages correlated with their ability to exploit low levels of cell surface CD4 for infection (12, 14). Determinants within and proximal to the CD4 binding site (CD4bs) were shown to modulate macrophage infectivity (3, 4, 5, 12, 13) and presumably acted by altering the avidity of the trimer for cell surface CD4. These determinants include residues proximal to the CD4 binding loop, which is likely the first part of the CD4bs contacted by CD4 (1). We also observed that macrophage-tropic R5 envelopes were frequently more resistant to the glycan-specific monoclonal antibody (MAb) 2G12 than were non-macrophage-tropic R5 Envs (13).Here, we investigated the envelope determinants of 2G12 sensitivity by using two HIV-1 envelopes that we used previously to map macrophage tropism determinants (4), B33 from brain and LN40 from lymph node tissue of an AIDS patient with neurological complications. While B33 imparts high levels of macrophage infectivity and is resistant to 2G12, LN40 Env confers very inefficient macrophage infection and is 2G12 sensitive (12-14).     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. ​(Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man₉GlcNAc₂ precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man₅GlcNAc₂ intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. ​(Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man₃GlcNAc₂ pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. ​(Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc₃Man₉GlcNAc₂; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man₅GlcNAc₂ (Fig. ​(Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man₅GlcNAc₂ substrate (19) (Fig. ​(Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. ​(Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. ​Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc₃Man₉GlcNAc₂ precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man₉GlcNAc₂ precursor into Man₅GlcNAc₂ or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. ​(Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man₅GlcNAc₂ substrate, preventing the formation of hybrid and complex carbohydrates (Fig. ​(Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man₅GlcNAc₂ rather than with the larger Man₉GlcNAc₂ precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man₅GlcNAc₂ we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120₃/gp41₃). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.Identification of conserved regions accessible on the HIV-1 envelope and design of immunogens that elicit broadly neutralizing antibodies against these sites continue to be major challenges in the development of an effective HIV-1 vaccine. The HIV-1 viral spike—composed of three exterior gp120 subunits and three transmembrane gp41 subunits—is highly protected, but a limited number of these conserved regions exist on the spike, identified primarily by the broadly neutralizing antibodies that target them. One region is quaternary in nature and appropriately formed only on the assembled viral spike (gp120₃/gp41₃). This region is targeted by a recently discovered (14) and fast expanding class of monoclonal antibodies (36, 40) that recognize epitopes with quaternary structural constraints, which are composed of portions of two gp120-variable loops, V2 and V3 (reviewed in reference 49). These quaternary structure-specific (or quaternary-specific) antibodies (also called quaternary-neutralizing epitope or “QNE” antibodies) are found in the sera of selected HIV-1-infected individuals who have broadly neutralizing serum antibodies (41); individual members of the class, however, vary greatly in their breadth of neutralization.Initial evidence for the existence of quaternary-specific antibodies arose in simian/human immunodeficiency virus-infected rhesus macaques and HIV-1-infected chimpanzees (6, 9, 13). Characterization of polyclonal sera from these infected animals suggested the presence of antibodies targeting a conformational epitope involving the variable loop regions of the gp120 viral envelope.Antibody 2909 was the first human monoclonal antibody against HIV-1 to be characterized as being specific for an epitope dependent on the quaternary interaction of envelope glycoproteins (14). It was identified by direct screening for neutralization activity against a pseudovirus derived from strain SF162 of HIV-1. It recognizes a quaternary epitope on the surface of native virions and infected cells but does not bind soluble gp120/gp140 envelope proteins or cell surface-expressed gp120 monomers (14, 20). Competition analysis and virological assays indicate that the 2909 epitope includes portions of the V2 and V3 loops of gp120 (14, 16), with the V2-V3 elements originating either from within a gp120 monomer or between gp120 protomers in the trimer context. Mapping of 2909 recognition identifies a particular anomaly in its recognition (16); neutralization by 2909 depends on the presence of a rare lysine at position 160 in the V2 loop rather than the conserved N-linked site of glycosylation found at this position in most HIV-1 isolates (providing a residue-specific explanation for the neutralization specificity of 2909 for the SF162 virus, which contains this rare lysine).Other strain-specific monoclonal antibodies like 2909 have been isolated from rhesus macaques infected with a chimeric simian/human immunodeficiency virus that contained an SF162 isolate-derived viral spike (SHIV_SF162P4) (36). These rhesus monoclonal antibodies exhibit properties similar to those of 2909 in their potent neutralization of SF162 and their recognition of V2-V3 only in the context of the functional viral spike (e.g., on virus particles) (36). Details from epitope mapping indicate that these rhesus antibodies and human antibody 2909 recognize overlapping epitopes, with some differences in requirements for V2 N-linked glycosylation (36).The somatically related human monoclonal antibodies, PG9 and PG16, were also identified by a direct screen for neutralization (40). They target a quaternary-specific V2-V3 epitope, but unlike 2909, they neutralize an extraordinary 70 to 80% of circulating primary HIV-1 isolates and appear to have some reactivity for monomeric gp120 (40). Much of their increased breadth of neutralization arises from their ability to recognize an N-linked glycan at position 160 in the V2 loop, a motif which is found in greater than 90% of HIV-1 group M isolates (25).Despite substantial differences in their neutralization breadth, antibodies 2909 and PG9/PG16 may be closely related. Notably, an N160K mutation in the V2 loop of typical primary HIV-1 isolates like YU2 and JR-FL can recover 2909 activity (16). Conversely, isolate SF162 can be converted to a PG9- and PG16-sensitive pseudovirus by the K160N mutation (40). Thus, a single N or K at position 160 appears to control much of the neutralization difference between 2909 and PG16. Together the results suggest that 2909 and PG9/PG16 antibodies recognize distinct immunotypes of a similar quaternary epitope.To gain insight into how antibodies achieve recognition of this epitope, we determined the crystal structure of the antigen-binding fragment (Fab) of 2909 at a 3.3-Å resolution and compared this structure to the previously determined structure of PG16 (31, 33). Mutational analysis was used to confirm structural hot spots, and chimeric analysis of domain swaps between 2909 and other quaternary-specific antibodies was used to refine assessments of functional similarity. By identifying structural features—shared between 2909 and PG16 but otherwise highly uncommon in antibodies—the results provide insight into conserved solutions by human antibodies for recognition of an important vaccine target on HIV-1.     

Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.     

A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Δpmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Δmnn1 deletion to the Δpmr1 background, ensuring the exposure of terminal α1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man₈GlcNAc₂ structures in Δoch1 Δmnn1 Δmnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies.The human immunodeficiency virus (HIV) has evolved numerous means to evade the humoral immune response, including a two-receptor mechanism for entry that recesses and protects highly conserved binding sites in the gp120 subunit of the viral envelope (Env) protein, trimerization of Env to further protect neutralizing epitopes readily exposed on the monomer, and rapid and continual mutation in the face of immune selective pressure (8, 9). Another highly effective defense mechanism is found in the extensive array of oligosaccharides covering gp120, with approximately 25 N-linked glycosylation sites per gp120 monomer (26). These glycans facilitate HIV type 1 (HIV-1) escape from immune surveillance by presenting immunologically “self” molecules with highly variable glycoforms that mask polypeptide epitopes along the “silent face” of gp120 (46, 49). Additionally, high-mannose-type N-linked glycans on gp120 have been implicated in inducing immunosuppressive responses from dendritic cells (DCs) (40), and in helping viral dissemination by binding to DCs through C-type lectins, such as DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing nonintegrin) (18, 33, 34). The high affinity of DC-SIGN for mannose structures on gp120 (29, 41), and evidence that DC-SIGN⁺ mucosal cells assist trans infection of permissive T cells, imply a key role for DC-SIGN in early HIV infection after sexual transmission (19).The high-mannose-type glycans of gp120 also represent a vulnerability for HIV-1. Mannose-binding lectins, such as cyanovirin N (16), actinohivin (12), and human mannose-binding protein (17), can interact with gp120 and inhibit HIV-1 infection in vitro. More critically for vaccine studies, high-mannose glycans are also the target of 2G12, one of the few broadly neutralizing monoclonal antibodies (MAbs) isolated from HIV-1-infected patients (36, 37, 42). The potency of this MAb stems from its unique epitope on the exposed and relatively conserved “silent face” of gp120, comprised of a cluster of terminal Manα1,2-Man residues on the D1 and D3 arms of up to three high-mannose glycans (10, 11, 36, 37). 2G12 is thought to have a high affinity for these gp120 glycans due to a unique heavy chain variable (V_H) domain-swapped configuration that forms a multivalent binding surface with a potential noncanonical binding site at the novel V_H/V_H interface in addition to the two conventional V_H/light chain variable (V_L) binding sites. This extended antigen binding surface is thought to allow 2G12 to interact with multiple clustered high-mannose glycans (11).Due to the broadly neutralizing activity of 2G12, the high-mannose glycans on gp120 have aroused interest in the design of glycoantigens that recapitulate the 2G12 epitope. Several such antigens have been created by using flexible linkers to cross-link natural or chemically synthesized high-mannose glycans to various molecular scaffolds, each showing that multivalency of high-mannose glycans is the key to higher 2G12 affinity (2, 25, 27, 43-45). An alternative approach is to express heterologous glycoproteins with natural high-mannose glycans able to support 2G12 binding (28, 38). The yeast Saccharomyces cerevisiae expresses many proteins with high densities of N-linked glycans, and the enzymes involved in its N-glycosylation pathway are easily manipulated to produce glycans with various high-mannose structures (3, 31). We previously showed that an engineered strain lacking the OCH1, MNN1, and MNN4 genes for carbohydrate-processing enzymes expressed at least four highly glycosylated proteins that supported 2G12 binding and that immunization of rabbits with whole yeast cells from this strain elicited antibodies that cross-reacted with the glycans of gp120 (28). Here, we describe a second approach to modify the glycosylation machinery of S. cerevisiae and the subsequent discovery of Pst1, a yeast glycoprotein able to bind MAb 2G12. We show that Pst1 displays increased 2G12 binding as the dominant glycans on the protein become more similar to the glycans on the 2G12 epitope of gp120. This form of Pst1, containing strictly Man₈GlcNAc₂ glycans, displayed high affinity for 2G12 and effectively blocked the interaction of gp120 with 2G12 and DC-SIGN. This identifies Pst1 as a candidate molecular scaffold for an effective presentation of the 2G12 epitope and as a potential immunogen to induce mannose-specific antibodies.     

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

Poxviruses produce complement regulatory proteins to subvert the host''s immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host''s immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement''s role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE''s regulatory capacity. These results suggest that EMICE''s role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.Poxviruses encode in their large double-stranded DNA genomes many factors that modify the immune system (30, 56). The analysis of these molecules has revealed a delicate balance between viral pathogenesis and the host''s immune response (2, 21, 31, 61). Variola, vaccinia, monkeypox, cowpox, and ectromelia (ECTV) viruses each produce an orthologous complement regulatory protein (poxviral inhibitor of complement enzymes [PICE]) that has structural and functional homology to host proteins (14, 29, 34, 38, 41, 45, 54). The loss of the regulatory protein resulted in smaller local lesions with vaccinia virus lacking the vaccinia virus complement control protein (VCP) (29) and in a greater local inflammatory response in the case of cowpox lacking the inflammation-modulatory protein (IMP; the cowpox virus PICE) (35, 45, 46). Additionally, the complete loss of the monkeypox virus inhibitor of complement enzymes (MOPICE) may account for part of the reduced mortality observed in the West African compared to Congo basin strains of monkeypox virus (12).The complement system consists of proteins on the cell surface and in blood that recognize and destroy invading pathogens and infected host cells (36, 52). Viruses protect themselves from the antiviral effects of complement activation in a variety of ways, including hijacking the host''s complement regulatory proteins or producing their own inhibitors (7, 8, 15, 20, 23). Another effective strategy is to incorporate the host''s complement regulators in the outermost viral membrane, which then protects the virus from complement attack (62). The extracellular enveloped virus (EEV) produced by poxviruses acquires a unique outer membrane derived from the Golgi complex or early endosomes that contain the protective host complement regulators (58, 62). Poxviruses have multiple infectious forms, and the most abundant, intracellular mature virions (IMV), are released when infected cells lyse (58). The IMV lacks the outermost membrane found on EEV and is sensitive to complement-mediated neutralization. The multiple strategies viruses have evolved to evade the complement system underscore its importance to innate and adaptive immunity (15, 36).The most well-characterized PICE is VCP (24-29, 34, 49, 50, 53, 55, 59, 60). Originally described as a secreted complement inhibitor (34), VCP also attaches to the surface of infected cells through an interaction with the viral membrane protein A56 that requires an unpaired N-terminal cysteine (26). This extra cysteine also adds to the potency of the inhibitor by forming function-enhancing dimers (41). VCP and the smallpox virus inhibitor of complement enzymes (SPICE) bind heparin in vitro, and this may facilitate cell surface interactions (24, 38, 50, 59). The coevolution of variola virus with its only natural host, humans, likely explains the enhanced activity against human complement observed with SPICE compared to the other PICEs (54, 64).Our recent work with ECTV, the causative agent of mousepox infection, demonstrated that the classical and alternative pathways of the complement system are required for host survival (48). The mouse-specific pathogen ECTV causes severe disease in most strains and has coevolved with its natural host, analogous to variola virus in humans (9). This close host-virus relationship is particularly important for evaluating the role of the complement system, given the species specificity of many complement proteins, receptors, and regulators (10, 47, 62). Additionally, the availability of complement-deficient mice permits dissection of the complement activation pathways involved. Naïve C57BL/6 mouse serum neutralizes the IMV of ECTV in vitro, predominately through opsonization (48). Maximal neutralization requires natural antibody, classical-pathway activation, and amplification by the alternative pathway. C3 deficiency in the normally resistant C57BL/6 strain results in acute mortality, similar to immunodeficiencies in important elements of the antiviral immune response, including CD8⁺ T cells (19, 32), natural killer cells (18, 51), and gamma interferon (33). During ECTV infection, the complement system acts in the first few hours and days to delay the spread of infection, resulting in lower levels of viremia and viral burden in tissues (48).This study characterized the PICE produced by ECTV, ectromelia virus inhibitor of complement enzymes (EMICE), and assessed its complement regulatory activity. Recombinant EMICE (rEMICE) decreased activation of both human and mouse complement. Murine cells produced EMICE at 4 to 6 h postinfection prior to the release of the majority of the complement-sensitive IMV from infected cells. rEMICE protected ECTV IMV from complement-mediated neutralization. Further, EMICE produced during natural infection inhibited complement deposition on infected cells by the alternative pathway. ECTV likely produces this abundance of EMICE to protect both the IMV and infected cells.     

Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC₅₀) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC₅₀ titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.Since the identification of human immunodeficiency virus type 1 (HIV-1) as the cause of AIDS, one of the greatest challenges has been the development of a vaccine that will prevent infection and/or ameliorate disease progression (38, 43). Although over 100 phase I, II, and III vaccine clinical trials of different candidates have been conducted all over the world, only a few candidates have advanced to efficacy testing and none has yet to show any benefit in prevention or control of HIV-1 (HIV Vaccine Database; www.iavi.org). In other viral diseases (such as polio, influenza, and measles), neutralizing antibodies are generated as part of either the natural immune response to infection or the response to immunization, and their role in protective immunity is well established (10, 12, 15, 22, 37, 42, 45, 47, 49, 52). For HIV-1, studies in animal models indicate that both broadly neutralizing antibodies and cell-mediated responses may be required to provide vaccine protection (7, 14, 16, 20, 29, 31, 33, 34, 39, 53). Unlike many other viruses, HIV-1 is highly variable, with multiple subtypes and recombinant forms circulating in different regions of the world. This high level of HIV-1 genetic variability, particularly in the envelope glycoproteins (gp120 and gp41), has been one of the greatest obstacles in development of a safe and effective HIV-1 vaccine and in particular in the elicitation of broadly neutralizing antibodies. In addition, HIV-1 has other mechanisms of immune escape preventing elicitation of broadly neutralizing antibodies, including the heavy glycosylation of the envelope glycoproteins, instability of such glycoproteins, and conformational masking of receptor-binding sites (6, 25, 32).Despite the enormous diversity of HIV-1, a relatively small number of broadly neutralizing monoclonal antibodies (bnMAbs) have been isolated, providing evidence that broad neutralization by single antibody specificities can be achieved (3-5, 8, 9, 17, 21, 23, 24, 29, 35, 36, 40, 41, 44, 50, 51, 55). Structures for such bnMAbs have been determined in complex with HIV-1 Env (26, 54) and provide starting points for the design of immunogens capable of eliciting broadly neutralizing antibodies. However, since there are only a few such bnMAbs, we established a global program as part of International AIDS Vaccine Initiative''s (IAVI''s) Neutralizing Antibody Consortium (6), aimed at screening HIV-1⁺ subjects with the goal of identifying individuals with broad and potent neutralizing activities as a potential source of novel bnMAbs, with an emphasis placed on individuals infected with non-clade B viruses. This paper describes the screening algorithm implemented to successfully identify HIV-1⁺ subjects with broadly neutralizing antibodies, including a subset of individuals termed “elite neutralizers.” These volunteers will be studied further to characterize the specificities of serum antibodies and will provide source materials for isolation of bnMAbs.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM⁻ cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.Cytokinesis in Escherichia coli and other gram-negative bacteria proceeds via the coordinated constriction of their envelope layers (outer membrane, inner membrane, and peptidoglycan [PG]) (12, 13, 34, 89). This coordination is achieved by a multi-protein division machine referred to as the septal ring or divisome (20). Assembly of the septal ring begins with the polymerization of the bacterial tubulin protein, FtsZ, into a ring structure just underneath the inner membrane at the prospective site of cell division (8). Once formed, this so-called Z-ring facilitates the recruitment of a number of essential and nonessential division proteins to the division site for the assembly of the trans-envelope divisome organelle (20).A major function of the cytokinetic machinery is to promote the synthesis of the PG layer that will eventually fortify the new poles of the developing daughter cells. PG is a polysaccharide polymer composed of repeating units of N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) linked by a β-1,4-glycosidic bond (46). Attached to the MurNAc sugar is a short peptide that is used to form cross-links between adjacent polysaccharide strands (46). Such cross-links allow for the construction of a cell-shaped PG meshwork that surrounds the cell membrane and protects it from osmotic rupture.A new wave of zonal PG synthesis is initiated at the division site during each cell cycle (23, 25, 72, 77, 91). Several of the major PG synthases called penicillin-binding proteins are components of the divisome organelle and play important roles in the synthesis of PG during division (7, 21, 62, 67, 73, 74, 80, 81, 88, 90). The septal PG layer produced by these and perhaps other components of the divisome is thought to be initially shared by the daughter cells (46). In gram-positive bacteria, this septal PG layer is typically split some time after the daughter cells have been compartmentalized by membrane fusion (11). In gram-negative bacteria, however, the septal PG layer is split shortly after it is formed to allow constriction of the outer membrane to closely follow that of the inner (cytoplasmic) membrane (12, 13, 34, 89). This gives rise to the characteristic constricted appearance of predivisional cells of E. coli and its relatives.PG hydrolysis is required to promote septal PG splitting and eventual daughter cell separation (87). E. coli, like many bacteria, encodes a vast array of factors with known or predicted PG hydrolase activity (at least 30 genes and 11 different protein families) (29, 31, 87). In most cases, the loss of individual PG hydrolase factors has little effect on growth and division, suggesting that there is significant functional overlap between the various hydrolases (87). This dearth of phenotypic information has consequently made it difficult to understand the physiological roles of PG hydrolases and identify the subset of these factors needed for septal PG splitting. An approach that has helped overcome this limitation in E. coli, however, has been the systematic deletion of all members of a particular PG hydrolase family from the genome (22, 44, 45, 63). Thus far, of all the families of PG hydrolases encoded by E. coli, the factors that play the predominant role in cell separation appear to be the LytC-type N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC (44, 45, 69). Loss of all three of these amidases results in a severe defect in cell separation and the formation of extremely long cell chains. This chaining phenotype can be exacerbated by the loss of members of other classes of PG hydrolases like the lytic transglycosylases or d,d-endopeptidases (44, 68). However, relative to strains defective for the amidases, mutants lacking multiple lytic transglycosylases or d,d-endopeptidases alone do not display significant chaining phenotypes in E. coli. These PG hydrolases therefore appear to be playing more of an ancillary role in cell separation.The LytM (lysostaphin/peptidase M23)-domain containing factors (referred to as LytM factors for convenience) are a widely distributed class of putative PG hydrolases that have been poorly characterized with regard to their role in PG biogenesis in E. coli and other bacteria (31). The most well-studied members of this family of factors, LytM and lysostaphin, are metallo-endopeptidases that cleave the pentaglycine cross-bridges found in staphylococcal PG (9, 30, 64). Based on this activity, other LytM factors are also likely to be PG hydrolases but with altered cleavage specificity because pentaglycine cross-bridges are only found among the staphylococci (75). Indeed, the LytM protein, gp13, from the Bacillus subtilis phage Φ29 was recently shown to be a d,d-endopeptidase that cleaves the meso-diaminopimelic acid-d-Ala cross-links of B. subtilis PG (17).E. coli encodes four factors with identifiable LytM-domains: EnvC, NlpD, YgeR, and YebA (29) (Fig. ​(Fig.1).1). Of the four, only EnvC has been studied in appreciable detail. EnvC⁻ mutants have a mild cell separation (chaining) defect when grown in medium containing salt and a severe division defect when grown at high temperatures in medium lacking salt (5, 42, 48, 71). In addition, purified EnvC protein was found to possess PG hydrolase activity using a gel-based zymogram assay, and an EnvC-green fluorescent protein (GFP) fusion exported to the periplasm via the Tat system was shown to be recruited to the division site (5). In all, these results support a model in which EnvC is targeted to the division site to participate directly in septal PG splitting and daughter cell separation.Open in a separate windowFIG. 1.Predicted domain structure of the E. coli LytM factors. Shown is a diagram depicting the predicted domain architecture of the four E. coli factors with identifiable LytM domains. Abbreviations: LytM, LytM domain; LysM, LysM PG-binding domain (29); CC, coiled coil; T, transmembrane domain; SS, signal sequence; SS*, lipoprotein signal sequence. The UniProtKB/Swiss-Prot accession numbers are as follows: EnvC (P37690), NlpD (P0ADA3), YebA (P0AFS9), and YgeR (Q46798).In the present study, we investigated the physiological role(s) of the entire set of E. coli LytM factors by generating mutant strains lacking all possible combinations of them. We found that, like the amidases, LytM factors play a critical role in daughter cell separation. Furthermore, studies of their subcellular localization revealed that NlpD is recruited to the division site along with EnvC, indicating that both of these LytM factors are likely to be participating directly in the septal PG splitting process. We also discovered that mutants lacking multiple LytM factors lyse more slowly and display an altered morphological response relative to wild-type (WT) cells when they are treated with ampicillin. This finding suggests that in addition to cell separation, LytM proteins play a role in the lytic mechanism of β-lactam antibiotics.     

Genetic,Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.It is now well established that protein glycosylation based on both N- and O-linked modifications occurs in bacterial species. In N-linked systems exemplified by the system in Campylobacter jejuni, large numbers of proteins that are translocated to the periplasm are glycosylated based on the presence of sequon elements and asparagine-targeting oligosaccharyltransferases related to those that operate in eukaryotes (21, 36, 69, 73). Two O-linked systems associated with covalent modification of type IV pilin subunits in pathogenic Neisseria species and in selected strains of Pseudomonas aeruginosa have been particularly well characterized (2, 16, 46-48, 54). The latter systems are remarkably similar to the N-linked system characterized in C. jejuni in that oligosaccharides are synthesized cytoplasmically as lipid-linked precursors that are then flipped into the periplasm. Protein-targeting oligosaccharyltransferases structurally related to the WaaL family of O-antigen ligases then transfer the oligosaccharides to protein substrates (2, 18, 49). The similarities between these N- and O-linked systems are perhaps best illustrated by genetic and functional interactions between components of the C. jejuni oligosaccharide biosynthetic machinery and elements of the neisserial pilin glycosylation pathway (2, 18). In contrast, the mechanisms operating in other bacterial O-linked systems are not completely understood yet, and there appears to be considerable diversity in the mechanisms of oligosaccharide synthesis, transfer of the glycan to the protein, and the cellular compartment in which glycan addition takes place. Prime examples of this diversity include the glycosylation of major subunits of S-layers (53), flagella (40), and type IV pili, as well as nonpilus adhesins, such as autotransporters (7, 51) and a family of serine-rich proteins identified in Gram-positive species (72). Recently, the pilin glycosylation system in the Gram-negative species Neisseria gonorrhoeae (the etiological agent of gonorrhea) was shown to be a general O-linked system in which a large set of structurally distinct periplasmic proteins undergo glycosylation (64). Likewise, a general O-linked glycosylation system targeting periplasmic and surface-exposed proteins has been documented in Bacteroides fragilis (19). In addition, an increasing number of lipoproteins in Mycobacterium tuberculosis have been found to be O glycosylated, and current evidence suggests that a single glycosylation pathway operates with these proteins (50).The large number of bacterial protein glycosylation systems strongly suggests that these systems are advantageous and affect fitness. In fact, mutants with mutations in the general glycosylation systems of C. jejuni and B. fragilis are defective in mucosal colonization, although the fundamental basis for the observations is unclear (19, 23). In some cases, defects in protein stability and trafficking have been documented. Examples of the latter have been reported for the Aida and Ag43 autotransporter adhesins of Escherichia coli and the serine-rich Fap1 streptococcal adhesin (11, 35, 72). In these cases, the glycosylation status appears to influence protein integrity along with intracellular or membrane trafficking events.Glycosylation may also influence protein structure and function or activity at the extracellular level. In the context of host-symbiont and host-pathogen interactions, bacterial cell surface polysaccharides and glycolipid glycans are well-established targets of both innate and adaptive immune responses (13, 61). However, the potential influence of protein-linked carbohydrate on immune recognition and signaling is only beginning to be investigated. Given the well-established effect of conjugating protein to carbohydrate on glycan-related immunogenicity, glycoproteins could be predicted to promote a robust T-cell-dependent antibody response directed toward glycan epitopes. In line with this, immunization of mice with O-glycosylated type IV pilin from P. aeruginosa strain 1244 (which bears a single repeat unit of the O antigen, the dominant component of its lipopolysaccharide) resulted in protection against challenge with immunological specificity for the O-polysaccharide (27). In addition, structural heterogeneity of carbohydrate modifications has been shown to affect the serospecificity of Campylobacter flagellins (41). With regard to innate immunity, the N-linked protein glycans of C. jejuni have been shown to influence interleukin-6 production by human dendritic cells via interaction with the macrophage galactose-type lectin (MGL) (62). Also, flagellin glycosylation of the phytopathogenic bacteria Pseudomonas syringae pv. glycinea and P. syringae pv. tomato appears to play an important role in hypersensitive cell death in nonhost plants and in host cell recognition (56, 57). Similarly, the flagellin glycosylation status in P. aeruginosa influences proinflammatory responses in human cell cultures (63).Studies of O-linked flagellar glycosylation in P. aeruginosa, C. jejuni, and a number of Gram-positive species have revealed considerable variability in genomic glycosylation islands (40). In addition to differences in gene content, some genes localized in these loci are subject to phase (on-off) variation involving slipped-strand mispairing events. Similar findings have been obtained for the O-linked glycosylation system in N. gonorrhoeae and a related system in Neisseria meningitidis (2, 4, 29, 48). These observations strongly suggest that protein-associated glycans are positively selected. However, attempts to elucidate the evolutionary processes impacting these systems are complicated by difficulties in connecting genotype with phenotype. For example, predicting enzymatic activities of components involved in glycan biosynthesis based on the sequence alone is notoriously difficult. Therefore, glycosylation-related functions are characterized best by using purified components in in vitro assays. Moreover, despite recent advances in mass spectrometric (MS) and nuclear magnetic resonance (NMR) technologies, glycoprotein structural analysis is still arduous, particularly when proteins are expressed at low levels. Thus, current methodologies are not optimized for studies of large numbers of strains and mutants.The broad-spectrum O-linked protein glycosylation system of N. gonorrhoeae is particularly well characterized with regard to the genetics of oligosaccharide biosynthesis, modification, and transfer to protein via the PglO/PglL oligosaccharyltransferase. As shown using strain N400, combined genetic and MS analyses, including interspecies complementation, have revealed that this system (designated the pgl [protein glycosylation] system) is remarkably similar to the N-linked system of C. jejuni with respect to the use of a peptide-proximal 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH) sugar and related biosynthetic pathways for generating lipid-linked glycan substrates (2, 18, 39). The lipid-linked DATDH sugar can be further converted successively into hexose (Hex)-DATDH disaccharide and Hex-Hex-DATDH trisaccharide forms by the PglA and PglE glycosyltransferases, respectively (2). The hexoses in both the di- and trisaccharide forms can also undergo O acetylation by the PglI enzyme (2, 70). As pglA, pglE, and pglI are each predicted to be subject to phase variation in some backgrounds, strains have the potential to express five distinct glycoforms (2, 4, 29, 48, 70). A similar system operates in N. meningitidis strain c311, although to date only pilin and the AniA nitrite reductase proteins have been shown to be glycosylated (37). Pioneering analyses of pilin from this strain identified a trisaccharide with a terminal alpha-1-4-linked digalactose moiety attached to DATDH (54). Interestingly, nearly one-half of N. meningitidis isolates are reported to have a unique allele of pglB designated pglB2 associated with synthesis of a proximal glyceramido-acetamido trideoxyhexose (GATDH) rather than DATDH (10). In addition, some strains of both N. gonorrhoeae and N. meningitidis have been reported to contain additional genes predicted to encode glycosyltransferases linked to the core locus that includes the pglF, pglB, pglC, and pglD genes (32, 48). Thus, it appears that the number of protein-associated glycans may be far greater than currently perceived. The genus Neisseria also includes a number of related species that colonize humans, including Neisseria lactamica, which is closely related to N. gonorrhoeae and N. meningitidis but is rarely associated with disease (24), as well as other, more divergent commensal species. An examination of recently available genome sequences of these nonpathogenic species revealed that they contain open reading frames (ORFs) whose products share high levels of amino acid identity with many of the protein glycosylation components found in N. gonorrhoeae and N. meningitidis and with many of the N. gonorrhoeae proteins targeted for glycosylation. However, protein glycosylation has not been documented in any of these species yet.Here, we developed a systematic approach for elucidating intra- and interstrain glycan diversity and its genetic basis in neisserial O-linked glycans by employing serotyping, mass spectrometric analyses, and genetically defined recombinant backgrounds. We then used these tools to demonstrate that protein-associated glycans are antigenically variable and that isolates of N. meningitidis and N. lactamica also exhibit broad-spectrum O-linked protein glycosylation.     

MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.Most bacteria have characteristic cell morphologies maintained during growth (67). The peptidoglycan (PG) component of the cell wall represents in most cases the physical support of various bacterial shapes. PG is a mesh-like polymeric macromolecule which opposes the osmotic pressure of the bacterial cytoplasm and prevents lysis in hypotonic growth environments (29). Isolated PG cell walls (sacculi) retain the shapes of the cells from which they originate while PG disruption causes the formation of osmotically labile spheroplasts, underscoring PG''s essential role in cell shape determination and cellular integrity maintenance. PG is composed of long glycan chains that are oriented roughly along the short axis of rod-shaped Gram-negative bacteria and that are connected by short peptide cross-links (21, 60). Bacterial growth and division necessitate the expansion and division of the PG cell wall, which requires the insertion of new PG material in the preexisting, covalently linked mesh (29). New PG synthesis requires two enzymatic reactions performed by penicillin-binding proteins (PBPs). Glycan chain synthesis is achieved by transglycosylation activity while cross-linkage of glycan chains to the existing mesh is achieved by transpeptidation activity (47). Class A PBPs, called bifunctional or bimodular PBPs (e.g., PBP1a and 1b of Escherichia coli), possess both transpeptidase and transglycosylase domains while class B PBPs, such as PBP2 and PBP3 of E. coli, can perform only transpeptidase reactions (47). Controlled degradation of the PG by cell wall hydrolases is necessary for incorporation of new PG material during growth. Tight coordination between PG synthesis and degradation is required to maintain the integrity of the mesh at all times (29).The bacterial cytoskeleton also plays a central role in cell shape determination and maintenance (7). MreB is a bacterial actin homolog that forms dynamic helical structures underneath the cytoplasmic membrane in most rod-shaped bacteria (8, 34, 37, 56). In some species, the spatial distribution of MreB varies during the cell cycle, changing from a helical/patchy localization pattern throughout the cell to a ring-like distribution near midcell (20, 22, 50, 58). MreB is required for rod shape maintenance as deletion of the MreB-encoding gene or depletion of MreB causes loss of rod shape and cell rounding (20, 22, 34, 63). Other proteins, including MreC, MreD, RodA, PBP2, and RodZ, function along with MreB to maintain rod shape as loss of their function also results in cell rounding (2, 5, 33, 48, 62). Among these rod-morphogenic proteins, only PBP2 has a known enzymatic function, being involved in PG synthesis as an elongation-specific transpeptidase; the others are membrane-spanning or integral membrane proteins (2, 5, 15, 48). The overall involvement of these morphogenetic proteins in rod shape maintenance has led to a model in which they are part of the elongase complex, a PG synthesizing machine that elongates the PG side wall (2, 5, 15, 48, 59). The elongase complex would include PG lytic enzymes and at least one bifunctional PBP required for glycan strand synthesis (15, 59). In Bacillus subtilis, MreB homologs were found to associate with the bifunctional PBP1 (36) and to regulate the localization of the PG hydrolase LytE (9). However, it is still unclear how MreB functions in the context of the proposed elongase complex to determine and maintain rod shape.It has been previously shown that repletion of MreB in lemon-shaped, MreB-depleted Caulobacter crescentus cells leads to the formation of cell filaments that present branches and ectopic stalks (64). To examine how MreB can drive de novo rod shape morphogenesis, we followed a similar strategy except that we used drug treatment to interfere with MreB function. The small molecule 3,4-dichlorobenzyl carbamimidothioate, also known as A22, has been shown to rapidly disrupt MreB localization in vivo and to induce growth-dependent rounding in several Gram-negative bacteria (23, 32, 41, 45, 52). Furthermore, genetic and biochemical experiments have shown that MreB is the direct molecular target of A22 and that A22 binds to MreB''s ATP-binding pocket, inducing a state with low affinity for polymerization (3, 23). As removal of A22 is followed within minutes by recovery of the normal MreB localization pattern (23), this drug represents a convenient tool for rapid and reversible inhibition of MreB function. However, A22 was found to inhibit the growth of an mreB deletion mutant of E. coli, suggesting that it can have MreB-independent toxic effects (35). In this study, we show that the toxicity of A22 varies with the drug concentration, culture medium conditions, and Gram-negative species tested. We identify a similarly potent but less toxic structural analog, MP265 (4-chlorobenzyl carbamimidothioate), as well as nontoxic concentrations and conditions for both A22 and MP265 that induce loss of rod cell morphology in C. crescentus. We also show that recovery of rod shape after drug removal is accompanied by intensive remodeling of PG morphology and composition.     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Role of N-Linked Glycosylation of the 5-HT2A Receptor in JC Virus Infection

JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR). The 5-HT_2AR contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT_2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT_2AR-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT_2AR to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT_2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT_2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT_2AR resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT_2AR-expressing cells. These data affirm the importance of 5-HT_2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT_2AR.The initial interaction between virus and host occurs via molecular interactions of viral attachment proteins and receptors on host cells. Therefore, receptor recognition is a critical host cell determinant and may play a key regulatory role in viral pathogenesis. The polyomavirus JC virus (JCV) is a ubiquitous human pathogen (21, 25, 32) that is initially subclinical yet establishes a persistent infection in the kidney (11). In immunosuppressed individuals JCV can become reactivated, leading to infection in the central nervous system (CNS) (13-15, 20), where the virus specifically targets glial cells, including astrocytes and the myelin-producing cells, oligodendrocytes (40, 48). JCV infection and cytolytic destruction of oligodendroglia cause the fatal disease progressive multifocal leukoencephalopathy (PML) (1, 22). The most common cause of PML is associated with human immunodeficiency virus (HIV) and AIDS (10, 23). However, in recent years PML has been reported in patients receiving immunosuppressive therapies for autoimmune diseases such as Crohn''s disease (44), multiple sclerosis (MS) (24, 26, 28, 47), systemic lupus erythematosus (5, 33), and rheumatoid arthritis (5, 19, 37). The prognosis of PML is bleak, as the disease progresses rapidly and usually proves fatal within 1 year of the onset of symptoms. While current treatment options for PML are limited (23), recent studies suggest that mirtazapine, a serotonin receptor antagonist, may be capable of slowing the progression of PML (6, 27, 45, 46).JCV has a nonenveloped, icosahedral capsid that encapsidates a circular double-stranded DNA (dsDNA) genome (39). JCV attachment to cells is mediated by an N-linked glycoprotein with either α(2,3)- or α(2,6)-linked sialic acid (16, 31), suggesting that N-linked glycosylation of cellular receptors is important for JCV infection. N-linked glycosylation is a posttranslational process by which oligosaccharides are added to asparagine residues, and this modification is important for protein processing, folding, expression, and function (43). Previous studies from our laboratory revealed that the JCV also requires the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR) to mediate JCV infection (18, 35, 38), while others report that JCV infection can occur in the absence of 5-HT_2AR (7, 8). 5-HT_2AR is a seven-transmembrane-spanning G-protein-coupled receptor that belongs to a large family of 5-HT serotonin receptors. 5-HT_2AR is abundantly expressed on cells in the brain (4), including glial cells (3), and in the kidney (4), which parallels the sites of JCV infection. N-linked glycosylation plays a key regulatory role in the function of serotonin receptors. Mutation of N-linked glycosylation sites in human 5-HT_3AR and 5-HT_5AR results in decreased expression at the plasma membrane, which is critical for receptor function (17, 34). N-linked glycosylation of murine 5-HT_3AR regulates plasma membrane targeting, ligand binding, Ca²⁺ flux, and receptor trafficking (36), suggesting that glycosylation is essential for expression and function of serotonin receptors.While previous studies have concluded that JCV utilizes an N-linked glycoprotein with α(2,3)-linked sialic acid (31) or α(2,6)-linked sialic acid (16) and 5-HT_2AR (18) to initiate infection in host cells, the mechanism(s) by which JCV engages its cellular receptors and the importance of receptor glycosylation remain unclear. 5-HT_2AR contains potential asparagine (N)-linked glycosylation sites, five of which are predicted to be expressed in the extracellular amino-terminal region, where they could be accessible to the virus (2). The goal of this study was to determine whether potential N-linked glycosylation sites expressed in 5-HT_2AR are required for JCV infection. We found that N-linked glycosylation of 5-HT_2AR is important for receptor expression but not necessary for JCV infection.