Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³⁶ in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³⁹INN sequence, which ablates the overlapping Asn¹⁴¹-Asn¹⁴²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu⁵⁹³, Trp⁵⁹⁶ and Lys⁶⁰¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.     

Global and Local Structural Properties of the Principal Neutralizing Determinant of the HIV-1 Envelope Protein gp120

Abstract

The model of spatial structure for the principal neutralizing determinant (PND) of the HIV-1 envelope protein gpl20 is proposed in terms of two-dimensional nuclear Overhauser effect (NOE) spectroscopy data. To build the model, the NMR-based theoretical conformational analysis of synthetic PND peptides of length 40, 24, and 12 residues is carried out. The modeling of the molecular spatial structures is performed by a new approach to research of conformationally mobile peptides using the algorithms of the restrained molecular mechanics method developed earlier. The following major conclusions are made based on the analysis of the simulated peptide conformations: i) there is not unique PND structure in solution, ii) there are seven different PND structures each of which agrees with the experimental data and stereochemical criteria used in computing its spatial model, iii) the PND is characterized by irregular conformation containing a number of reverse turns, iv) all of the selected conformations are conserved in the Gly-Pro-Gly-Arg-Ala-Phe stretch, the most provable viral immunodominant epitope. These data allow to suppose that binding properties of this site are determined by the structural motif which forms the conformation of a double β-turn and appears common for all hexapeptide structures.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

A short segment of the HIV-1 gp120 V1/V2 region is a major determinant of resistance to V1/V2 neutralizing antibodies

Antibody PG9 is a prototypical member of a class of V1/V2-directed antibodies that effectively neutralizes diverse strains of HIV-1. We analyzed strain-specific resistance to PG9 using sequence and structural information. For multiply resistant strains, mutations in a short segment of V1/V2 resulted in gain of sensitivity to PG9 and related V1/V2 neutralizing antibodies, suggesting both a common mechanism of HIV-1 resistance to and a common mode of recognition by this class of antibodies.     

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.     

Structure of an HIV-2 gp120 in Complex with CD4

HIV-2 is a nonpandemic form of the virus causing AIDS, and the majority of HIV-2-infected patients exhibit long-term nonprogression. The HIV-1 and HIV-2 envelope glycoproteins, the sole targets of neutralizing antibodies, share 30 to 40% identity. As a first step in understanding the reduced pathogenicity of HIV-2, we solved a 3.0-Å structure of an HIV-2 gp120 bound to the host receptor CD4, which reveals structural similarity to HIV-1 gp120 despite divergence in amino acid sequence.     

Molecular Recognition of CCR5 by an HIV-1 gp120 V3 Loop

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.     

Mutations in both gp120 and gp41 Are Responsible for the Broad Neutralization Resistance of Variant Human Immunodeficiency Virus Type 1 MN to Antibodies Directed at V3 and Non-V3 Epitopes

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative α-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.     

The V1/V2 loop of HIV-1 gp120 is necessary for Tat binding and consequent modulation of virus entry

Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells.     

A broad spectrum anti-HIV inhibitor significantly disturbs V1/V2 domain rearrangements of HIV-1 gp120 and inhibits virus entry

Inhibition of human immunodeficiency virus (HIV) entry into target human cells is considered as a critical strategy for preventing HIV infection. Conformational shifts of the HIV-1 envelope glycoprotein (gp120) facilitates the attachment of the virus to target cells, therefore gp120 remains an attractive target for antiretroviral therapy development. Compound 18A has been recently identified as a broad-spectrum anti-HIV inhibitor. It was proposed that 18A disrupts rearrangements of V1/V2 region in gp120; however, the precise mechanism by which 18A interferes with the inherent motion of V1/V2 domain remains obscure. In this report, we elaborate on the binding mode of compound 18A to the closed conformation of a soluble cleaved gp120 and further examine the dynamic motion of V1/V2 region in both gp120 and the gp120–18A complex via all-atom molecular dynamics simulations. In this work, comparative molecular dynamic analyses revealed that 18A makes contact with Leu179, Ile194, Ile424, Met426 W427, E370 and Met475 in the main hydrophobic cavity of the unliganded gp120 and disrupts the restructuring of V1/V2 domain observed in apo gp120. The unwinding of α1 and slight inversion of β2 in gp120 leads to the shift of VI/V2 domain away from the V3 N-terminal regions and toward the outer domain. Stronger contacts between Trp425 and Trp112 rings may contribute to the reduced flexibility of α1 observed upon 18A binding thereby inhibiting the shifts of the V1/V2 region. Binding of 18A to gp120: (1) decreases the overall flexibility of the protein and (2) inhibits the formation a gp120 conformation that closely ressembles a CD4-bound-like conformation. Information gained from this report not only elaborates on important dynamic features of gp120, but will also assist with the future designs of potent gp120 inhibitors as anti-HIV.     

Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC₅₀) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.Immunoglobulin molecules have multiple binding paratopes for antigens; for example, those for IgG1 are bivalent and those for IgM are dodecavalent. It is obvious that multivalent binding is required for the distinct mechanism of neutralization by cross-linking multiple virions to form virus aggregates (reviewed in references 7 and 67). Despite the potential of antibodies for multivalent binding, structural evidence indicates that neutralizing antibodies often bind to an individual virion in a monovalent fashion (19, 20, 27, 29, 50, 53; reviewed in references 12 and 22). Bivalent binding of an antibody to a virion has been documented with clear structural evidence in only one case, in which monoclonal antibodies (MAbs) 17-IA and 8F5 bind to virions of human rhinovirus 14 (HRV14) and HRV2 (19, 43). Even in this unique case, binding bivalency appears to contribute to the neutralization potency of 17-IA but not to that of 8F5 (19, 42, 43). Moreover, these MAbs bind to two hydrophobic canyon structures formed by viral proteins VP1 and VP2 and not to antigenic epitopes within individual viral capsid protomers; thus, this case may represent an exception to the common form of antibody/antigen interactions in which the antibodies bind to individual antigens. Therefore, it is not clear what role antibody-binding multivalency plays in antibody-mediated neutralization of viruses at the level of interaction between antibody molecules and individual virions.The binding affinity of an antibody to its target is defined by intrinsic affinity and avidity (reviewed in reference 16). Intrinsic affinity is the force of monovalent binding between an antibody paratope and an antigenic epitope, often measured by binding a Fab fragment to an antigen. Avidity is the additive or synergistic force of engaging multiple antibody paratope/antigen epitope pairs between one antibody and one antigen. In other words, avidity is a functional consequence of antibody-binding multivalency. The effect of avidity on affinity is readily demonstrated in biochemical reactions such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), in which high-density antigenic sites are available without distinct spatial restrictions. It is commonly assumed that both affinity and avidity have functional consequences in antibody-mediated neutralization of viruses (reviewed in references 7 and 67). At the level of individual virions, the contribution of antibody-binding avidity to neutralization efficiency is often based on two types of experiments. In one, results from a side-by-side comparison between an antibody and its Fab fragment are often reported as evidence supporting a role of antibody-binding multivalency in virus neutralization. However, the interpretation of this type of experiment is complicated by the size difference between an antibody and a Fab fragment, since steric hindrance is a major mechanism of neutralization (reviewed in references 6 and 23). In a second type of experiment, a correlation between neutralization efficiency and the ability of the antibody/virus complex to resist chemical stress without dissociation in the presence of a high concentration of salt in solution is interpreted to support a contributing effect from antibody-binding avidity to neutralization efficiency (2, 21, 36, 49, 51). Data from this type of experiment are limited mostly to measuring binding affinity that is below the affinity required for virus neutralization. Furthermore, these studies often do not distinguish between avidity effects caused by an antibody binding to two (or more) epitopes on one antigen or to multiple epitopes from different molecules on the virion. Therefore, like the situation with antibody-binding multivalency, it remains unclear whether binding avidity contributes to antibody-mediated neutralization of viruses at the level of individual virions.The envelope glycoproteins (Envs) of human immunodeficiency virus type 1 (HIV-1) exist on the virion or cell surface as trimers of gp120 and gp41 heterodimers (13, 30, 62, 65). High-resolution structural information for a native HIV-1 Env trimer is critically important for understanding the function of HIV-1 Envs as well as for guiding the development of an effective immunogen to elicit broad and potent neutralizing antibody responses. X-ray crystal structures of the gp41 ectodomain fragments in the postfusion conformation have been resolved; however, a high-resolution structure of gp41 in the prefusion conformation is still unavailable and likely will be more informative for understanding the function of HIV-1 Env trimers (9, 47, 52). Two X-ray crystal structures of the gp120 core in both the CD4-liganded and unliganded conformations have been solved, but the biological meanings of these structures, especially how they are related to the native, functional Env trimer, are still being debated (10, 26). Several low-resolution structures of the Env trimers from HIV-1 or the closely related simian immunodeficiency virus (SIV) have been determined using cryoelectron microscopy (cryo-EM) tomography (4, 30, 62, 64, 65, 66). The predicted structures for the Env trimer are in general quite different between the two studies, and the difference is particularly dramatic around the gp41 membrane-proximal external region (MPER). A high-resolution structure of the native HIV-1 Env trimer is needed to resolve these differences. In the meantime, a distinctive standard needs to be developed for evaluating the biological relevance of structural information of an HIV-1 Env trimer.Our previous studies of the stoichiometry of antibody-mediated neutralization of HIV-1 Env indicated that MAbs b12, 2G12, and 2F5 neutralize by a stoichiometry designated T=1, i.e., one antibody binds to and neutralizes one HIV-1 Env trimer (57). Furthermore, when an artificial epitope (FLAG) was inserted in the V4 region of HIV-1 gp120, an epitope-specific anti-FLAG MAb achieved neutralization by the mechanism of steric hindrance (37, 61). Using the well-defined 2F5 neutralizing epitope as a model system (35, 39, 45), we constructed HIV-1 Env proteins carrying one 2F5 epitope in the gp120 V4 region and another 2F5 epitope in the gp41 MPER. Here, we investigated whether binding bivalency leads to enhancement in neutralization efficiency. By studying the detailed requirement for binding bivalency, we also probed the structure of the native, functional HIV-1 Env trimer, aiming to establish a standard that can be employed to evaluate the biological relevance of structural information on the HIV-1 Env trimer.     

Modification of Delivery System Enhances MHC Nonrestricted Immunogenicity of V3 Loop Region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2^b, H-2^d, H-2^k and H-2^S, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P<0.05 to P< 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

Effect of Lysine to Arginine Mutagenesis in the V3 Loop of HIV-1 gp120 on Viral Entry Efficiency and Neutralization

HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R₉R₁₀R₁₁ showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K₉K₁₀K₁₁ viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data.     

Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor

Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.     

A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV_cpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV_cpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.     

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).     

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.