Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes.     

HIV病毒包膜蛋白gp120与卡介苗感染人巨噬细胞的实验研究

目的:比较研究HIV病毒包膜蛋白gp120与卡介苗分别及共同感染人巨噬细胞,对人巨噬细胞的破坏能力及诱导巨噬细胞产生一氧化氮(NO)能力的差异性.方法:gp120与卡介苗(BCG)分别及共同感染人巨噬细胞后,于不同时间点采用MTT法检测巨噬细胞存活率,利用硝酸还原酶法检测细胞培养上清液中NO的含量.结果:gp120与BCG分别及共同感染人巨噬细胞,均可降低巨噬细胞的存活率,但gp120与卡介苗共同感染巨噬细胞,其存活率降低更为显著(P＜0.05);gp120与BCG均可激活人巨噬细胞合成和释放NO,而gp120与BCG共同感染组激活人巨噬细胞合成和释放NO的量明显低于BCG感染组(P＜0.05).结论:gp120感染巨噬细胞可影响巨噬细胞抗微生物的活性,可增强卡介苗对巨噬细胞的破坏作用.     

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

CD4⁺ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4⁺ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4⁺ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.The development of an effective vaccine against HIV has been hampered by an incomplete understanding of the correlates of protection against the virus. It is generally accepted that a robust antibody response and cytotoxic T-lymphocyte (CTL) response are required to control the disease and to prevent progression to AIDS (2, 17, 19, 20, 36, 38-42). Both of these arms of the immune system require help from CD4⁺ helper T cells (1, 27, 48). However, several important aspects of the CD4⁺ helper T-cell response remain poorly defined; these include the factors that determine epitope immunodominance in the CD4⁺ T-cell response, the relationship of specificity in the CD4⁺ T-cell response to specificity in the antibody and CD8⁺ responses, and the investment made by HIV (or any pathogen) to control the CD4⁺ T-cell response.Previous studies of mice showed that antigen structure modulates antigen processing and presentation of CD4⁺ helper T-cell epitopes (3-6, 9, 10, 23, 24, 43). Immunodominant CD4⁺ helper T-cell epitopes raised in response to immunization with the HIV envelope glycoprotein gp120 were found adjacent to flexible loops between elements of secondary structure (10). This was rationalized by the fact that flexible loops more readily conform to protease active sites and therefore are preferentially cleaved by proteases during antigen processing (10, 14, 15). Helper T-cell epitopes of gp120 in humans infected with HIV were also found flanking flexible loops (30). Dominant epitopes were located in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops (30). These results suggested that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in both mice and humans.In reviewing previous studies mapping the helper T-cell response to gp120, we noted a marked absence of CD4⁺ T-cell responses to regions of the outer domain that coincided with the locations of highly conserved disulfide bonds (Fig. ​(Fig.1).1). Disulfide bonds have previously been shown to interfere with presentation of nearby helper T-cell epitopes (13, 26). Thus, we hypothesized that disulfide bonds stabilized these regions of the protein, protecting them from proteolysis. This resulted in the exclusion of these regions from presentation to helper T cells. We further hypothesized that the deletion of these disulfide bonds would result in the production of new helper T-cell epitopes by creating localized regions of flexibility that could now be processed and presented to T cells. The creation of new helper T-cell epitopes could also potentially lead to changes in the antibody response.Open in a separate windowFIG. 1.Gaps in helper T-cell epitope frequency in the outer domain of HIV gp120 coincide with the locations of disulfide bonds. The graph illustrates the frequencies of responses by residue for the combined profiles from immunized BALB/c and CBA mice (gray area) and for a group of seven HIV-infected human subjects (black line) (10, 30).For the present work, we constructed three disulfide-bond variants of gp120 by replacing paired cysteines in the outer domain with alanines. Characterization of the variants revealed that the proteins were structurally distinct from one another and from wild-type gp120. Groups of 10 BALB/c mice immunized with these proteins produced patterns of helper T-cell responses that were very different from each other and from that of a group of 10 BALB/c mice immunized with wild-type gp120. In general, the T-cell response was reduced in mice immunized with the variant proteins. For one of the variants, anti-gp120 antibody titers were increased and exhibited CD4-blocking activity.     

HIV-1 Envelope Glycoprotein Variable Loops Are Indispensable for Envelope Structural Integrity and Virus Entry

HIV-1 envelope (Env) glycoprotein is a trimer of heterodimer of gp120 and gp41, and derives from a trimeric glycoprotein precursor, gp160. Gp120 contains five conserved regions that are interspersed with 5 variable loop regions (V1–V5). Env variations in variable loop length and amino acid composition may associate with virus pathogenesis, virus sensitivity to neutralizing antibodies (nAbs) and disease progression. To investigate the role of each variable loop in Env function, we generated a panel of JRFL gp160 loop deletion mutants and examined the effects of each loop deletion on Env expression, Env cell surface display and Env-mediated virus entry into permissive cells. We found that deletion of V1 and V2 (ΔV1V2), or loop D (ΔlpD) abolished virus entry, the same effect as deletion of V3 (ΔV3), while deletion of V3 crown (ΔV3C) significantly enhanced virus assembly and entry. We further found that deletion of V4 (ΔV4) or V5 (ΔV5), or replacement of V4 or V5 with flexible linkers of the same lengths knocked out the receptor and coreceptor binding sites in gp120, but significantly enhanced the exposure of the N-trimer structure and the membrane proximal external region (MPER) in gp41. Although deletion of V4 or V5 did not affect Env expression, they negatively affected Env cell surface display, leading to the failure in virus assembly and subsequent entry. Taken together, we found that Env variable loops were indispensable for Env structural integrity and virus entry. Our findings may have implications for development of HIV-1 vaccine immunogens and therapeutics.     

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion

Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.     

Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion,Incorporation, and Infectivity

The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.Human immunodeficiency virus type 1 (HIV-1) infection is initiated by fusion of the viral membrane with that of the target cell and is mediated by the viral envelope glycoprotein (Env). HIV-1 Env, a type 1 membrane-spanning glycoprotein, is a trimeric complex composed of three noncovalently linked heterodimers of gp120, the receptor-binding surface (SU) component, and gp41, the membrane-spanning, transmembrane (TM) component (12, 26, 44, 45). The gp120 and gp41 glycoproteins are synthesized as a precursor gp160 glycoprotein, which is encoded by the env gene. The gp160 precursor is cotranslationally glycosylated and, following transport to the trans-Golgi network, is cleaved into the mature products by a member of the furin family of endoproteases (45). Mature Env proteins are transported to the plasma membrane, where they are rapidly endocytosed or incorporated into virions (5, 33, 43). Recent evidence suggests that endocytosis and intracellular trafficking of Env is required for its interaction with Gag precursors and for efficient assembly into virions (20).HIV-1 Env molecules function as quasistable “spring-loaded” fusion machines. Recent studies have suggested that several regions of gp120 are reoriented following CD4 binding so that a planar “bridging sheet,” which forms the binding site for the coreceptor (CCR5 or CXCR4), can form (6, 7). Coreceptor binding is necessary for additional conformational changes in gp41 and for complete fusion (3). The gp41 monomer has three subdomains, an ectodomain, a membrane-spanning domain (MSD), and a cytoplasmic domain (39). The ectodomain of gp41, which mediates membrane fusion, is composed of a fusion peptide, two heptad repeats, and a tryptophan-rich membrane-proximal external region. Following the binding of gp120 to the CD4 receptor and the CCR5/CXCR4 coreceptor, conformational changes are induced in Env that result in the exposure of the gp41 fusion peptide (32). This peptide inserts into the target cell membrane, allowing gp41 to form a bridge between the viral and cellular membranes. Interaction of the heptad repeats to form a six-helix bundle then brings the target and viral membranes together, allowing membrane fusion to occur (24).While heptad repeat regions 1 and 2 in the N-terminal ectodomain play key roles in Env-mediated fusion by bringing the viral and cell membranes into close proximity, an important function of gp41 is to anchor the glycoprotein complex within the host-derived viral membrane (18). The precise boundaries of the HIV-1 MSD have not been clearly defined; however, the MSD is one of the most conserved regions in the gp41 sequence. Based on the initial functional studies of HIV-1, the MSD of Env was defined as a stretch of 25 predominantly hydrophobic amino acids that span residues K681 to R705 in the NL4-3 sequence (14, 16, 18). These residues were suggested to cross the viral membrane in the form of an alpha helix, the length of which is approximately equal to the theoretical depth of a membrane bilayer. A major caveat of this model is that it places a basic amino acid residue (R694) into the hydrophobic center of the lipid bilayer. While some transmembrane proteins do contain charged amino acid residues in their MSDs, it is normally considered to be energetically unfavorable without some mechanism to neutralize the charge (8, 13). Point mutation studies have yielded varying results, but in general, substitution of K681 is detrimental to fusion and infectivity while mutation of R694 or R705 has only a limited effect on these activities (16, 29). On the other hand, accumulating data argue for a different intramembrane structure of the HIV-1 MSD. Serial small deletions (3 amino acid residues) in the region between R694 and R705 showed normal cell-cell fusion, although larger deletions were detrimental (29), suggesting that, with respect to the biological functions of the Env glycoprotein, the length of this region is more important than its amino acid conservation.Previous C-terminal-truncation studies of simian immunodeficiency virus (SIV) Env (19, 41) suggested that the entire 27-amino-acid region is not required for the biological function of the protein. In the case of SIV, only the 15 apolar amino acids flanked by K689 and R705 (equivalent to K681 and R694 in HIV) and 6 additional amino acids (for a total of 23 amino acids) were required for near-wild-type (WT) fusion (19, 41). Two subsequent residues were required (total, 25 amino acids) for virus-cell entry and infectivity, while a length of 21 amino acid residues was sufficient for SIV Env to be incorporated into viral particles. These results led to a basic amino acid “snorkeling” model for the SIV MSD (41). In this model, the lysine and arginine (NL4-3 equivalents of K681 and R694) are buried in the lipid bilayer, while their long side chains are proposed to extend outward to the membrane surface and present the positively charged amino groups to the negatively charged head groups of the lipid bilayers. Applied to HIV-1 MSD, this model predicts a hydrophobic intramembrane core of only 12 amino acid residues (compared to 15 amino acid residues in the SIV MSD) between K681 and R694. The hydrophobic region C-terminal to K681 is not sufficient to effectively anchor the protein, since mutation of R694 to a stop codon yielded a nonfunctional protein that appeared to be retained in the endoplasmic reticulum (11). This contrasts with truncation experiments with the vesicular stomatitis virus (VSV) G glycoprotein, which have shown that a region of 12 hydrophobic amino acids flanked by basic residues is sufficient to anchor the protein in the membrane (1).In order to understand if the “snorkeling” model is applicable to the HIV-1 MSD, we constructed a series of nonsense mutants with HIV-1 gp41 truncated in single-amino-acid steps at the C terminus from residue R707 to residue R694. For each mutant Env, we determined the membrane stability, fusogenicity, and ability to mediate infectivity. The results of these studies suggest that the 12-residue “core” (36) plus three subsequent hydrophobic amino acids is the minimal anchor domain for HIV-1 Env, as well as the minimal sequence to mediate cell-cell fusion. In contrast to SIV Env, HIV-1 Env requires the entire 25-amino-acid region from K681 to R707 to mediate near-WT incorporation and infectivity.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 β2, β19, β20, and β21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.The entry of human immunodeficiency virus type 1 (HIV-1) is mediated by the viral envelope glycoproteins (9, 79). The HIV-1 envelope glycoproteins are synthesized as an ∼850-amino acid precursor, which trimerizes and is posttranslationally modified by carbohydrates to create a 160-kDa glycoprotein (gp160). The gp160 envelope glycoprotein precursor is proteolytically processed in the Golgi apparatus, resulting in a gp120 exterior envelope glycoprotein and a gp41 transmembrane envelope glycoprotein (16, 17, 66, 76). In the mature HIV-1 envelope glycoprotein trimer, the three gp120 subunits are noncovalently bound to three membrane-anchored gp41 subunits (32).HIV-1 entry involves the binding of gp120 in a sequential fashion to CD4 and one of the chemokine receptors, CCR5 or CXCR4 (1, 8, 15, 18, 25, 36). CD4 binding triggers the formation of an activated intermediate that is competent for binding to CCR5 or CXCR4 (29, 69, 73, 78). These chemokine receptors are G protein-coupled, 7-transmembrane segment receptors with relatively short N termini. The choice of chemokine receptors is dictated primarily by the sequence of a gp120 region, the third variable (V3) loop, that exhibits variability among HIV-1 strains and becomes exposed upon CD4 binding (4, 8, 10, 33, 37, 38, 49, 59, 75). X-ray crystal structures of CD4-bound HIV-1 gp120 have revealed that the gp120 “core” consists of a gp41-interactive inner domain, a surface-exposed and heavily glycosylated outer domain, and a conformationally flexible bridging sheet (38, 43, 79). In the CD4-bound state, the V3 loop projects 30 Å from the gp120 core, toward the chemokine receptor (38). The V3 loop in these structures consists of three elements: (i) conserved antiparallel β strands that contain a disulfide bond at the base of the loop; (ii) a conformationally flexible stem; and (iii) a conserved tip (37, 38). During the virus entry process, the base of the gp120 V3 loop and elements of the bridging sheet interact with the CCR5 N terminus, which is acidic and contains sulfotyrosine residues (12-14, 23, 24). Sulfotyrosine 14 of CCR5 is thought to insert into a highly conserved pocket near the V3 base, driving further conformational rearrangements that result in the rigidification of the V3 stem (37). The conserved β-turn at the tip of the V3 loop, along with some residues in the V3 stem, is believed to bind the “body” of CCR5, i.e., the extracellular loops and membrane-spanning helices. CCR5 binding is thought to induce further conformational changes in the HIV-1 envelope glycoproteins, leading to the fusion of the viral and target cell membranes by the gp41 transmembrane envelope glycoproteins.CCR5 binding involves two points of contact with the gp120 V3 loop: (i) the CCR5 N terminus with the V3 base and (ii) the CCR5 body with the V3 tip and distal stem (12-14, 23, 24, 37, 38). The intervening V3 stem can tolerate greater conformational and sequence variation, features that might decrease HIV-1 susceptibility to host antibodies (30). Despite amino acid variation, the length of the V3 loop is well conserved among naturally occurring group M (major group) HIV-1 strains (30, 42). This conserved length may be important for aligning the two CCR5-binding elements of the V3 loop. In addition to allowing optimal CCR5 binding, the conserved V3 length and orientation may be important for CCR5 binding to exert effects on the conformation of the HIV-1 envelope glycoproteins. We examine here the consequences of introducing extra amino acid residues into the V3 stem. The residues were introduced either into both strands of the V3 loop, attempting to preserve the symmetry of the structure, or into one of the strands, thereby kinking the loop. The effects of these changes on assembly, stability, receptor binding, and the membrane-fusing capacity of the HIV-1 envelope glycoproteins were assessed. In addition to effects on chemokine receptor binding, unexpected disruption of gp120-gp41 association was observed. Further investigation revealed a conserved patch in the tip of the V3 loop that is important for the association of gp120 with the trimeric envelope glycoprotein complex, as well as for chemokine receptor binding. Apparently, the V3 loop and adjacent gp120 structures contribute to the stability of the trimer in the unliganded HIV-1 envelope glycoproteins. These structures are known to undergo rearrangement upon CD4 binding, suggesting their involvement in receptor-induced changes in the virus entry process.     

Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP₁ and GP₂, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.     

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.     

Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.     

The Protein Tyrosine Kinase p56lck Is Required for Triggering NF-κB Activation upon Interaction of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 with Cell Surface CD4

We have previously shown that NF-κB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559–5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-κB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56^lck as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56^lck, or both molecules, we provide direct evidence that expression of CD4 and p56^lck is required for HIV-1-induced NF-κB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-κB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56^lck indicates the requirement for a functional CD4-p56^lck complex.     

CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides—estimated at three to four per virion—is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S- and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.Hepatitis B virus (HBV) remains a major public health concern worldwide, affecting more than 350 millions of chronically infected individuals. Since the discovery of HBV, substantial information has been gathered on the viral replication cycle, but our understanding of the viral entry mechanism remains limited, and the identity of the receptor(s) for HBV is still unknown (15). HBV displays a very narrow host range, which is likely determined at viral entry by a highly specific interaction between the HBV envelope proteins and receptors at the surface of human hepatocytes. The envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (S-HBsAg) are membrane-spanning glycoproteins that differ from each other by the size of their N-terminal ectodomain (21). L-HBsAg contains a N-terminal pre-S1, central pre-S2, and C-terminal S domains. M-HBsAg is shorter than L-HBsAg in lacking pre-S1, whereas S-HBsAg consists of the S domain only (Fig. ​(Fig.1).1). Envelope protein synthesis occurs at the endoplasmic reticulum (ER) membrane. Empty subviral particles (SVPs) assemble from aggregates at a pre-Golgi membrane and exit the cell through the secretory pathway (36). Assembly of mature HBV virions requires, in addition to S-HBsAg, the presence of L-HBsAg as a matrix protein for nucleocapsid envelopment (6). Recent findings indicate that HBV virions and SVPs follow distinct pathways for budding: the late endosomal multivesicular bodies (MVBs) for HBV virions, and the MVB-independent secretory pathway for SVPs (26, 28, 46). The HBV envelope proteins can also package the hepatitis delta virus (HDV) ribonucleoprotein (RNP), in case of HBV/HDV coinfection (5, 45), leading to the formation of HDV virions. Whether HDV uses the SVP secretion pathway rather than an MVB-dependent route is uncertain.Open in a separate windowFIG. 1.Schematic representation of HBV envelope proteins. The topology of the L-, M-, and S-HBsAg proteins at the viral membrane is represented. The pre-S2 domain of L- and M-HBsAg, and the determinants of viral entry, pre-S1 and AGL, are indicated. The M-HBsAg protein, represented in gray, is dispensable for infectivity. The myristic acid (Myr) linked to the L-HBsAg N terminus is indicated (closed box). Subdomains 2-48 and 49-75 of the pre-S1 infectivity determinant are indicated. Open boxes represent transmembrane regions in the S domain.L-HBsAg, but not M-HBsAg, is crucial to infectivity of both HBV and HDV particles (13, 31, 41, 42). L-HBsAg contains a major infectivity determinant located between amino acid residues 2 and 75 of its N-terminal pre-S1 domain (4, 30), including a myristoyl anchor linked to glycine-2 (1, 8, 18), a putative receptor binding site (RBS) between positions 2 and 48, and a domain of unknown function between amino acids 49 and 75. To date, the most compelling evidence that pre-S1 mediates receptor binding comes from studies demonstrating that myristoylated synthetic peptides specific for the N-terminal 2-to-48 pre-S1 domain can bind to hepatocyte plasma membranes and block infection in vitro (3, 16, 17) and in vivo (37). Beside pre-S1, a second determinant was recently identified in the antigenic loop (AGL) borne by the three HBV envelope proteins (Fig. ​(Fig.1).1). The AGL participation in viral entry was first established in the HDV model (23) and more recently directly in the HBV model (39). Interestingly, serine substitutions for the AGL cysteine residues, which prove detrimental to the conserved immunodominant “a” determinant, could also block viral entry. Note that the “a” determinant consists in conformational epitopes, which elicit highly neutralizing antibodies (22). Infectivity and the “a” determinant were also lost when virions were treated with membrane-impermeable inhibitors of thiol/disulfide isomerization (2). These findings clearly established a correlation between the AGL cysteine disulfide bonds network, the conformation of the “a” determinant, and infectivity. Hence, the strict conservation of the “a” determinant among all HBV genotypes is related to the AGL function at viral entry. The AGL determinant may operate in association with, or independently of pre-S1, in binding to receptors at the early step of entry and/or in the mechanism of envelope disassembly postentry.In the present study, we investigated the pre-S1 determinant by performing transcomplementation experiments between mutants of 3 pre-S1 subelements: the myristoyl anchor, subdomain 2-48, and subdomain 49-75. We analyzed the activity of the AGL determinant in the S- or L-HBsAg background (S- and L-AGL, respectively), and we examined the effect of introducing increasing amounts of infectivity-deficient pre-S1, or AGL, in the virion''s envelope on infectivity.     

Linkage of Amino Acid Variation and Evolution of Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein (Subtype B) with Usage of the Second Receptor

To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains. This suggests that residue 440 in the C4 region, which is close to the V3 loop in the three-dimensional structure, is critical in determining which second receptor is used. Analysis of codon frequency suggests that, in almost all cases, the difference at residue 440 between basic amino acids in the R5 strains and nonbasic amino acids in the X4 strains could be due to a single nucleotide change. These findings predict that the evolutionary changes in amino acid residue 440 may be correlated with evolutionary changes in the V3 loop. One possibility is that a change in electric charge at residue 440 compensates for a change in electric charge in the V3 loop. The amino acid polymorphism at position 440 can be useful to predict the cell tropism of a strain of HIV-1 subtype B.     

Both Neutralization Resistance and High Infectivity Phenotypes Are Caused by Mutations of Interacting Residues in the Human Immunodeficiency Virus Type 1 gp41 Leucine Zipper and the gp120 Receptor- and Coreceptor-Binding Domains

Neutralization resistance of human immunodeficiency virus type 1 (HIV-1) is a major impediment to vaccine development. We have found that residues of HIV-1 MN strain in the C terminus of gp120 and the leucine zipper (LZ) region of gp41 viral envelope proteins interact cooperatively to determine neutralization resistance and modulate infectivity. Further, results demonstrate that this interaction, by which regions of gp120 are assembled onto the LZ, involves amino acid residues intimately related to those which participate in the binding of the envelope to its receptor and coreceptor. Variations in this critical assembly structure determine the concordant, interdependent evolution of increased infectivity efficiency and neutralization resistance phenotypes of the envelopes. The results elucidate important structure-function relationships among epitopes that are important targets of vaccine development.