Identification of an N-linked glycosylation in the C4 region of HIV-1 envelope gp120 that is critical for recognition of neighboring CD4 T cell epitopes

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.     

Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response

The envelope (Env) glycoprotein of human immunodeficiency virus (HIV) contains 24 N-glycosylation sites covering much of the protein surface. It has been proposed that one role of these carbohydrates is to form a shield that protects the virus from immune recognition. Strong evidence for such a role for glycosylation has been reported for simian immunodeficiency virus (SIV) mutants lacking glycans in the V1 region of Env (J. N. Reitter, R. E. Means, and R. C. Desrosiers, Nat. Med. 4:679-684, 1998). Here we used recombinant vesicular stomatitis viruses (VSVs) expressing HIV Env glycosylation mutants to determine if removal of carbohydrates in the V1 and V2 domains affected protein function and the generation of neutralizing antibodies in mice. Mutations that eliminated one to six of the sites for N-linked glycosylation in the V1 and V2 loops were introduced into a gene encoding the HIV type 1 primary isolate 89.6 envelope glycoprotein with its cytoplasmic domain replaced by that of the VSV G glycoprotein. The membrane fusion activities of the mutant proteins were studied in a syncytium induction assay. The transport and processing of the mutant proteins were studied with recombinant VSVs expressing mutant Env G proteins. We found that HIV Env V1 and V2 glycosylation mutants were no better than wild-type envelope at inducing antibodies neutralizing wild-type Env, although an Env mutant lacking glycans appeared somewhat more sensitive to neutralization by antibodies raised to mutant or wild-type Env. These results indicate significant differences between SIV and HIV with regard to the roles of glycans in the V1 and V2 domains.     

Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses

Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.     

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.     

Location-Specific, Unequal Contribution of the N Glycans in Simian Immunodeficiency Virus gp120 to Viral Infectivity and Removal of Multiple Glycans without Disturbing Infectivity

One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.     

Infectivity and neutralization of simian immunodeficiency virus with FLAG epitope insertion in gp120 variable loops

A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 mug/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.     

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

Virion envelope content, infectivity, and neutralization sensitivity of simian immunodeficiency virus

A truncating E767stop mutation was introduced into the envelope glycoprotein of simian immunodeficiency virus (SIV) strain SIV239-M5 (moderately sensitive to antibody-mediated neutralization and lacking five sites for N-linked carbohydrate attachment) and strain SIV316 (very sensitive to neutralization, with eight amino acid changes from the neutralization-resistant parental molecular clone, SIV239). The truncating mutation increased Env content in virions, increased infectivity, and decreased sensitivity to antibody-mediated neutralization in both strains. However, the magnitude of the effect on infectivity and neutralization sensitivity differed considerably between the two strains. In the context of strain SIV239-M5, truncation increased Env content in virions approximately 10-fold and infectivity in a reporter cell assay 24-fold. The truncated SIV239-M5 was only slightly more resistant to neutralization by polyclonal monkey sera and by monoclonal antibodies than SIV239-M5 with a full-length envelope glycoprotein. In the context of strain SIV316, truncation increased infectivity a dramatic 480-fold, while envelope content in virions was increased only about 14-fold. This dramatic increase in infectivity cannot be simply explained by the increase in envelope content and is likely due to an increase in inherent infectivity, i.e., infectivity per spike, that results from truncation. The truncated SIV316 was extremely resistant to antibody-mediated neutralization. In fact, it was not neutralized by any of the antibodies tested. When increasing amounts of SIV316 envelope glycoprotein (full length) were provided in trans to SIV316, infectivity was increased and sensitivity to neutralization was decreased, but to nowhere near the degree that was obtained when truncated SIV316 envelope glycoprotein was used. Truncated forms of SIV239 and SIV239-M5 required higher levels of soluble CD4 for inhibition of infection than their nontruncated forms; truncated SIV316 did not. Our results suggest that envelope content in SIV virions, infectivity, and resistance to antibody-mediated neutralization can be increased not only by truncation of the cytoplasmic domain but also by provision of excess envelope in trans. The striking increase in infectivity that results from truncation in the context of SIV316 appears to be due principally to an increase in inherent infectivity per spike.     

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.     

Mutational analysis of N-linked glycosylation sites of Friend murine leukemia virus envelope protein.

The roles played by the N-linked glycans of the Friend murine leukemia virus envelope proteins were investigated by site-specific mutagenesis. The surface protein gp70 has eight potential attachment sites for N-linked glycan; each signal asparagine was converted to aspartate, and mutant viruses were tested for the ability to grow in NIH 3T3 fibroblasts. Seven of the mutations did not affect virus infectivity, whereas mutation of the fourth glycosylation signal from the amino terminus (gs4) resulted in a noninfectious phenotype. Characterization of mutant gene products by radioimmunoprecipitation confirmed that glycosylation occurs at all eight consensus signals in gp70 and that gs2 carries an endoglycosidase H-sensitive glycan. Elimination of gs2 did not cause retention of an endoglycosidase H-sensitive glycan at a different site, demonstrating that this structure does not play an essential role in envelope protein function. The gs3- mutation affected a second posttranslational modification of unknown type, which was manifested as production of gp70 that remained smaller than wild-type gp70 after removal of all N-linked glycans by peptide N-glycosidase F. The gs4- mutation decreased processing of gPr80 to gPr90, completely inhibited proteolytic processing of gPr90 to gp70 and Pr15(E), and prevented incorporation of envelope products into virus particles. Brefeldin A-induced mixing of the endoplasmic reticulum and parts of the Golgi apparatus allowed proteolytic processing of wild-type gPr90 to occur in the absence of protein transport, but it did not overcome the cleavage defect of the gs4- precursor, indicating that gs4- gPr90 is resistant to the processing protease. The work reported here demonstrates that the gs4 region is important for env precursor processing and suggests that gs4 may be a critical target in the disruption of murine leukemia virus env product processing by inhibitors of N-linked glycosylation.     

Neutralizing Capacity of Monoclonal Antibodies That Recognize Peptide Sequences Underlying the Carbohydrates on gp41 of Simian Immunodeficiency Virus

Extensive glycosylation of the envelope spikes of human and simian immunodeficiency virus (HIV and SIV) is an important factor for the resistance of these viruses to neutralization by antibodies. SIVmac239 gp41 has three closely spaced sites for N-linked carbohydrate attachment. Rhesus macaques experimentally infected with mutant versions of SIVmac239 lacking two or three of these carbohydrate sites developed strong serum reactivity against mutated peptide sequences at the site of these glycosylations, as well as high titers of neutralizing activity to the mutant viruses (E. Yuste et al., J. Virol. 82:12472–12486, 2008). However, whether antibodies that recognize these underlying peptides have neutralizing activity has not been directly demonstrated. Here we describe the isolation and characterization of three gp41-specific monoclonal antibodies (4G8, 6G8, and 7D6) from one of these mutant-infected monkeys. All three antibodies reacted with mutant gp41 from viral particles and also with peptides corresponding to mutated sequences. Slight differences in peptide specificities were observed among the three antibodies. Sequence analysis revealed that the heavy chains of all three antibodies were derived from the same germ line heavy-chain segment (IGHV4-59*01), but they all had very different sequences in complementarity-determining region 3. The light chains of all three antibodies were very closely related to one another. All three antibodies had neutralizing activity to mutant viruses deficient in gp41 carbohydrate attachment, but they did not neutralize the parental SIVmac239. These results demonstrate unambiguously that antibodies with specificity for peptide sequences underlying gp41 carbohydrates can effectively neutralize SIV when these carbohydrates are absent. However, the presence of these gp41 carbohydrates effectively shields the virus from antibodies that would otherwise neutralize viral infectivity.     

Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage,Infectivity, and Immune Response

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein complex (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies (asparagine to glutamine, asparagine to alanine, and serine/tyrosine to alanine mutants) to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd (N89), 5th (N119), or 8th (N365) glycosylation motif. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd (N89Q) or 8th (N365Q) glycan completely inhibited the transduction efficiency of pseudotyped particles. We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st (N79Q), 3rd (N99Q), 5th (N119Q), or 6th (N167Q) glycan significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st (N79Q), 2nd (N89Q), 3rd (N99Q), 4th (N109Q), 5th (N119Q), 6th (N167Q), or 9th (N373Q) glycan enhanced the proportion of CD8+ effector T cells. Deletion of specific glycan improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine.     

Removal of N-linked glycosylation sites in the V1 region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.     

The Influence of N-Linked Glycans on the Molecular Dynamics of the HIV-1 gp120 V3 Loop

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.     

Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities

Simian immunodeficiency virus (SIV) of macaques isolate SIVmac239 is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, a property that it shares with most primary isolates of human immunodeficiency virus type 1 (HIV-1). This resistance is important for the ability of the virus to persist at high levels in vivo. To explore the physical features of the viral envelope complex that contribute to the neutralization-resistant phenotype, we examined a panel of SIVmac239 derivatives for sensitivity to neutralization by a large collection of monoclonal antibodies (MAbs). These MAbs recognize both linear and conformational epitopes throughout the viral envelope proteins. The variant viruses included three derivatives of SIVmac239 with substitutions in specific N-linked glycosylation sites of gp120 and a fourth variant that lacked the 100 amino acids that encompass the V1 and V2 loops. Also included in this study was SIVmac316, a variant of SIVmac239 with distributed mutations in env that confer significantly increased replicative capacity in tissue macrophages. These viruses were chosen to represent a broad range of neutralization sensitivities based on susceptibility to pooled, SIV-positive plasma. All three of these very different kinds of mutations (amino acid substitutions, elimination of N-glycan attachment sites, and a 100-amino-acid deletion spanning variable loops V1 and V2) dramatically increased sensitivity to neutralization by MAbs from multiple competition groups. Thus, the mutations did not simply expose localized epitopes but rather conferred global increases in neutralization sensitivity. The removal of specific N-glycan attachment sites from V1 and V2 led to increased sensitivity to neutralization by antibodies recognizing epitopes from both within and outside of the V1-V2 sequence. Surprisingly, while most of the mutations that gave rise to increased sensitivity were located in the N-terminal half of gp120 (surface subunit [SU]), the greatest increases in sensitivity were to MAbs recognizing the C-terminal half of gp120 or the ectodomain of gp41 (transmembrane subunit [TM]). This reagent set and information should now be useful for defining the physical, structural, thermodynamic, and kinetic factors that influence relative sensitivity to antibody-mediated neutralization.     

Identification of Replication-Competent Strains of Simian Immunodeficiency Virus Lacking Multiple Attachment Sites for N-Linked Carbohydrates in Variable Regions 1 and 2 of the Surface Envelope Protein

Carbohydrates comprise about 50% of the mass of gp120, the external envelope glycoprotein of simian immunodeficiency virus (SIV) and human immunodeficiency virus. We identified 11 replication-competent derivatives of SIVmac239 lacking two, three, four, or five potential sites for N-linked glycosylation. These sites were located within and around variable regions 1 and 2 of the surface envelope protein of the virus. Asn (AAT) of the canonical N-linked glycosylation recognition sequence (Asn X Ser/Thr) was changed in each case to the structurally similar Gln (CAG or CAA) such that two nucleotide changes in the codon would be required for reversion. Replication of one triple mutant (g456), however, was severely impaired. A revertant of the g456 mutant was recovered from CEMx174 cells with a Met-to-Val compensatory substitution at position 144, 2 amino acids upstream of attachment site 5. Thus, a debilitating loss of sites for N-linked glycosylation can be compensated for by amino acid changes not involving the Asn-X-Ser/Thr consensus motif. These results provide a framework to begin testing the hypothesis that carbohydrates form a barrier that can limit the humoral immune responses to the virus.     

N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV(+)-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.     

Importance of B-cell responses for immunological control of variant strains of simian immunodeficiency virus

The properties of three variants of cloned simian immunodeficiency virus strain 239 (SIV239) were compared. One strain (M5) lacked five sites for N-linked carbohydrate attachment in variable regions 1 and 2 (V1 and V2) of the gp120 envelope protein, one strain (DeltaV1-V2) completely lacked V1 and V2 sequences, and another (316) had nine mutations in the envelope that impart high replicative capacity for tissue macrophages. All three strains were capable of significant levels of fusion independent of CD4, and all three were considerably more sensitive to antibody-mediated neutralization than the parent strain from which they were derived. Upon experimental infection of rhesus monkeys, these three variant strains replicated to viral loads at peak height around day 14 that were indistinguishable from or only slightly less than those observed in monkeys infected with the parental SIV239 strain. Viral loads at the set point 20 to 50 weeks after infection, however, were more than 400- to 10,000-fold lower with the variant strains. Depletion of B cells around the time of infection with M5 resulted in less effective immunological control and much higher viral loads at the set point in two of three monkeys. The differences between SIV239 infection, where there is not effective immunological control, and SIVM5 infection, where there is effective immunological control, cannot be easily explained by differences in the inherent replicative capacity of the viruses; rather, they are more readily explained by differences in the effectiveness of the antibody response. These results suggest that resistance of SIV239 to antibody-mediated neutralization is very important for evading effective immunological control, for allowing continuous viral replication, for maintenance of moderate-to-high viral loads at set point, and for disease progression.