Toward Effective HIV Vaccination: INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY*

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421–433 and 288–306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V_H) domain framework (FR) residues. Substitution of the FR cavity V_H Lys-19 residue by an Ala residue resulted in attenuated binding of the 421–433 region peptide probe. The CDRs and V_H FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V_H1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421–433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.Induction of neutralizing antibodies (Abs)² via adaptive immune processes is the cornerstone of vaccination against microbial antigens. The antigen-binding site is mostly formed by the complementarity determining regions (CDRs) of the light and heavy chain variable domains (V_L and V_H domains). Vaccine-induced adaptive Ab responses entail sequence diversification of Ab V domains expressed within the B cell receptor (BCR) complex, selective noncovalent antigen binding to the high affinity BCR mutants, and proliferation of the mutant B cell clones. No HIV vaccine is available. The surface of HIV is studded with noncovalently associated oligomers of gp120 complexed to gp41. HIV infection and experimental HIV vaccination attempts induce robust Ab responses to the immunodominant epitopes of gp120, which are structurally divergent in various HIV strains responsible for infection in different parts of the world. Abs to such epitopes express strain-specific neutralization (1, 2), i.e. they neutralize the HIV strain from which the immunogen was isolated but not strains genetically heterologous to the immunogen.The gp120 site responsible for binding host CD4 receptors (CD4BS) is structurally more conserved. Precise conformational details of the CD4BS expressed on the HIV surface are not available, but crystallography suggests a large, discontinuous determinant composed of regions distant from each other in the linear protein sequence (3, 4). The 421–433 peptide region is essential for CD4 binding by gp120, suggested by contacts in the crystallized complex and loss of CD4 binding function by site-directed mutagenesis in this region (5, 6). The 421–433 region is a member of a small group of microbial polypeptide sites recognized selectively by Abs produced by the immune system without prior infection by the microbe (preimmune Abs) (7–9). Such sites are designated B cell “superantigens” (SAgs) because of their selective and widespread recognition by the comparatively conserved framework regions (FRs) of Ab V domains (10, 11). Noncovalent SAg binding by preimmune Abs, however, is characterized by low-to-moderate binding strength (12). Most gp120-binding preimmune Abs from humans without infection display poor or no HIV neutralizing activity (13). Patients with the autoimmune disease lupus and no HIV infection produce increased amounts of Abs to the 421–433 CD4BS region (14). A single chain Fv (scFv; V_L and V_H domains linked by a flexible peptide) from the lupus Ab repertoire that binds the 421–433 region reversibly neutralizes genetically diverse strains of HIV (15). Following completion of the noncovalent binding step, certain Abs can hydrolyze polypeptides via nucleophilic attack on carbonyl groups (16–21). The proteolytic reaction imparts improved antigen inactivation potency to Abs (22). We reported the neutralization of HIV by secretory IgA from humans without infection, an Ab class distinguished by the ability to catalyze the hydrolysis of gp120 selectively because of initial noncovalent recognition of the 421–433 CD4BS region (13).The conserved character of the CD4BS in genetically diverse HIV strains renders it suitable as a vaccine target. The CD4BS, however, is poorly immunogenic. Traditional immunization methods do not stimulate the adaptive synthesis of neutralizing Abs to the 421–433 region or other CD4BS epitopes. Neutralizing Abs that bind the CD4BS are found in the blood of a subset of patients after years of HIV infection, but the target epitope is not identified, and Ab response is weak (23, 24). Certain monoclonal Abs (mAbs) that bind the CD4BS expressed by purified monomer gp120 do not neutralize HIV appreciably or display limited ability to neutralize genetically diverse HIV strains (25, 26). The CD4BS is a flexible structure expressed in differing conformational states by monomer gp120 and the native gp120 oligomers of the virus (27–30). Moreover, the process of binding CD4 may induce movements within the CD4BS (31). Reproducing the native CD4BS conformation in experimental vaccine candidates has been difficult. A CD4BS mimetic of the epitope recognized by a well known anti-CD4BS neutralizing mAb (clone b12) did not induce the synthesis of neutralizing Abs (32). Polyclonal Abs raised by immunization with synthetic peptides spanning the 421–433 CD4BS region neutralized laboratory-adapted, coreceptor CXCR4-dependent HIV strains inconsistently (33–35). Neutralization of coreceptor CCR5-dependent strains responsible for initiating most HIV infections was not studied. Importantly, small synthetic peptides are often more flexible than the corresponding native protein segments. Inducing a traditional adaptive immune response in which the Ab CDRs develop binding specificity for the peptide immunogen therefore does not ensure recognition of the native 421–433 CD4BS region (35, 36). From mutagenesis and sequence identity studies, the gp120-binding site of preimmune Abs, in contrast, is composed mainly of the V_H domain FR1 and FR3 (10, 11, 37). As certain preimmune Abs express HIV neutralizing activity attributable to recognition of the 421–433 region (13), the FR-dominated site must recognize the native state of this CD4BS epitope expressed on the viral surface.There is, however, substantial difficulty in amplifying and improving the subset of preimmune Abs with HIV neutralizing activity for vaccination against the virus; SAg binding to Ab FRs fails to stimulate adaptive B cell differentiation and synthesis of specific IgG class Abs (38, 39). Indeed, the binding at the FRs may even lead to premature death of the B cells (12, 40). The SAg character of the 421–433 CD4BS epitope is therefore predicted to render it hypoimmunogenic with respect to the adaptive synthesis of neutralizing Abs following infection or traditional vaccination procedures.We reported previously the induction of nucleophilic Abs by covalent immunization with full-length gp120 and a gp120 V3 peptide containing strongly electrophilic phosphonate groups (41–43). The electrophile reacts covalently with BCRs (44), resulting in adaptively strengthened nucleophilic reactivity coordinated with specific noncovalent recognition of gp120. The Abs obtained by covalent immunization formed very stable immune complexes with HIV resulting from pairing of Ab nucleophiles with the naturally occurring electrophilic groups of gp120 (e.g. the backbone and side chain carbonyls, see Refs. 42, 43). A minority of the Abs proceeded to catalyze the hydrolysis of gp120, aided by water attack on the covalent acyl-Ab complex (41). Here we report the neutralization of HIV strains heterologous to the full-length electrophilic gp120 immunogen (E-gp120) by mAbs with binary CD4BS and V3 loop recognition capability. We also present models that explain synthesis of the mAbs in response to immunization with E-gp120.     

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.     

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed.The HIV-1 envelope glycoprotein complex (Env),³ a class I viral fusion protein, is responsible for viral attachment to CD4⁺ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1–3). The entry process of these viruses is an attractive target for therapeutic intervention.The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4, 5). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6, 7). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1, 8–12). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry.Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13–17). The HIV-1 fusion inhibitor T20 (enfuvirtide (Fuzeon)) has been approved for clinical use. T20 mimics HR2 and can bind to HR1, thereby preventing the formation of the six-helix bundle (Fig. 1) (18–21). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared with the first-generation peptide T20 (22–25). Recently, a series of more potent third-generation fusion inhibitors were designed (26, 27). These include T2635, which has an improved helical structure that increases stability and activity against both wild type (WT) HIV-1 and fusion inhibitor resistant variants.Open in a separate windowFIGURE 1.Schematic of the gp41 ectodomain. HR1 and HR2 are represented as cylinders, and position 38 in HR1 is indicated. Residues Gln-142, Asn-145, Glu-146, and Leu-149, which interact with residue 38, are underlined in the HR2 sequence. HR2-based peptide fusion inhibitors are shown underneath. Mutations introduced in T1249^mut and T2635^mut are bold and underlined. Numbering is based on the sequence of HXB2 gp41.Both the in vitro and in vivo selection of resistance has been described for T20 (28–33) and T1249 (23, 34–36). Resistance is often caused by mutations in the HR1 binding site of the fusion inhibitor. In particular, substitutions at positions 36 (G36D/M/S), 38 (V38A/W/M/E), and 43 (N43D/K) of gp41 can cause resistance. Strikingly, substitutions at position 38 can cause resistance to both T20 and T1249, but distinct amino acid substitutions are required. At position 38 only charged amino acids (V38E/R/K) cause resistance to T1249 (35). Surprisingly, none of the known T20 and T1249 resistance mutations at position 38 affect the susceptibility to the third generation inhibitor T2635.We hypothesized that the use of HIV-1 as a model system could provide a more detailed understanding of resistance to fusion inhibitors. We, therefore, analyzed the effect of all 20 amino acids at resistance hotspot 38 on Env function, viral fitness, biochemical properties of gp41, and resistance to the fusion inhibitors. From the results we can propose four resistance mechanisms that differ in the way the drug-target interaction is affected at the molecular level. Furthermore, we can deduce general principles on the mechanisms of resistance against fusion inhibitors and the requirements for effective antiviral drugs.     

Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.Infection of cells by human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with not only CD4 but also a second cell surface molecule, termed a coreceptor (reviewed in reference 19). Coreceptor usage varies significantly among different HIV-1 isolates, although all known coreceptors are members of the G-protein-coupled chemokine receptor family of seven-membrane-spanning receptors. The primary coreceptor used by non-syncytium-inducing, macrophage-tropic (M-tropic) HIV-1 isolates, which constitute the majority of primary isolates, is CCR-5 (1, 6, 8, 12, 27). In contrast, syncytium-inducing, T-cell-line-adapted (T-tropic) HIV-1 isolates predominantly use CXCR-4 as a coreceptor (13). Other chemokine receptors utilized by a small percentage of generally dualtropic HIV-1 isolates include CCR-2b and CCR-3 (6, 11). The importance of two orphan chemokine receptors, termed Bonzo/STRL33 and BOB/GPR15, in infection by HIV-1 remains to be established, although these proteins were recently shown to serve as coreceptors for several simian immunodeficiency virus and HIV-2 isolates (2, 9). The critical importance of CCR-5 for infection by primary, M-tropic HIV-1 isolates, however, has been highlighted by the finding that a small percentage of humans lack a functional CCR-5 gene and as a result appear highly, although not completely, resistant to infection by HIV-1 (17, 22). Importantly, primary T cells derived from such individuals are refractory to infection by M-tropic HIV-1 isolates in vitro (17, 22, 27), thus demonstrating that CCR-5 is the physiologically relevant coreceptor for the majority of primary isolates.At present, relatively little is known about how the viral envelope and coreceptor interact, although it appears clear that interaction is dependent upon a prior conformational shift induced by binding of the envelope gp120 subunit to CD4 (24, 26). This in turn is believed to lead to the formation of a ternary complex, consisting of gp120, coreceptor, and CD4, on the surface of the target cell (15, 24, 26). It is unknown how this protein complex then induces the fusion of the viral and host cell membranes, although the envelope gp41 subunit is believed to play a critical role at this stage.An important unresolved question is the identity of the amino acid residues in gp120 and the coreceptor that interact during infection. However, it is well established that HIV-1 tropism, and hence coreceptor usage, is largely controlled by a small number of residues located in the envelope V3 loop (6, 14, 23, 25). Efforts to identify residues in the CCR-5 coreceptor involved in mediating infection have thus far largely focused on the functional analysis of chimeric receptors generated with human CCR-5 (hCCR-5) and a chemokine receptor lacking coreceptor function, such as the murine CCR-5 homolog (mCCR-5) (3, 5, 20, 21). These studies have led to three major conclusions. Firstly, the residues in hCCR-5 involved in mediating HIV-1 infection are diffuse, being located on at least three of the four extracellular domains of CCR-5. Secondly, these residues are functionally redundant, so that several distinct regions of hCCR-5 can suffice independently to confer coreceptor function when substituted into mCCR-5. Lastly, different HIV-1 envelope proteins interact differently with CCR-5, such that CCR-5 residues important for mediating fusion by one envelope protein may be largely irrelevant to the interaction of CCR-5 with a second envelope protein. Overall, these data demonstrate that the envelope–CCR-5 interaction is likely to be highly complex and to involve the interaction of multiple residues in both proteins.As noted above, the mCCR-5 chemokine receptor, despite extensive sequence similarity to hCCR-5, fails to function as an HIV-1 coreceptor (3, 5, 20). Therefore, it is apparent that one or more of the 20 extracellular residues that differ between mCCR-5 and hCCR-5 must contribute to the interaction with the HIV-1 envelope protein. Using mutational analysis in the context of chimeric mCCR-5/hCCR-5 receptors, we have now identified several residues, located in three of the four extracellular domains of hCCR-5, that play roles in mediating infection by HIV-1.     

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

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

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.Human immunodeficiency virus type 2 (HIV-2) infection affects 1 to 2 million individuals, most of whom live in India, West Africa, and Europe (17). HIV-2 has diversified into eight genetic groups named A to H, of which group A is by far the most prevalent worldwide. Nucleotide sequences of Env can differ up to 21% within a particular group and by over 35% between groups.The mortality rate in HIV-2-infected patients is at least twice that of uninfected individuals (26). Nonetheless, the majority of HIV-2-infected individuals survive as elite controllers (17). In the absence of antiretroviral therapy, the numbers of infected cells (39) and viral loads (36) are much lower among HIV-2-infected individuals than among those who are HIV-1 infected. This may be related to a more effective immune response produced against HIV-2. In fact, most HIV-2-infected individuals have proliferative T-cell responses and strong cytotoxic responses to Env and Gag proteins (17, 31). Moreover, autologous and heterologous neutralizing antibodies (NAbs) are raised in most HIV-2-infected individuals (8, 32, 48, 52), and the virus seems unable to escape from these antibodies (52). As for HIV-1, the antibody specificities that mediate HIV-2 neutralization and control are still elusive. The V3 region in the envelope gp125 has been identified as a neutralizing target by some but not by all investigators (3, 6, 7, 11, 40, 47, 54). Other weakly neutralizing epitopes were identified in the V1, V2, V4, and C5 regions in gp125 and in the COOH-terminal region of the gp41 ectodomain (6, 7, 41). A better understanding of the neutralizing determinants in the HIV-2 Env will provide crucial information regarding the most relevant targets for vaccine design.The development of immunogens that elicit the production of broadly reactive NAbs is considered the number one priority for the HIV-1 vaccine field (4, 42). Most current HIV-1 vaccine candidates intended to elicit such broadly reactive NAbs are based on purified envelope constructs that mimic the structure of the most conserved neutralizing epitopes in the native trimeric Env complex and/or on the expression of wild-type or modified envelope glycoproteins by different types of expression vectors (4, 5, 29, 49, 58). With respect to HIV-2, purified gp125 glycoprotein or synthetic peptides representing selected V3 regions from HIV-2 strain SBL6669 induced autologous and heterologous NAbs in mice or guinea pigs (6, 7, 22). However, immunization of cynomolgus monkeys with a subunit vaccine consisting of gp130 (HIV-2BEN) micelles offered little protection against autologous or heterologous challenge (34). Immunization of rhesus (19, 44, 45) and cynomolgus (1) monkeys with canarypox or attenuated vaccinia virus expressing several HIV-2 SBL6669 proteins, including the envelope glycoproteins, in combination with booster immunizations with gp160, gp125, or V3 synthetic peptides, elicited a weak neutralizing response and partial protection against autologous HIV-2 challenge. Likewise, vaccination of rhesus monkeys with immunogens derived from the historic HIV-2ROD strain failed to generate neutralizing antibodies and to protect against heterologous challenge (55). Finally, baboons inoculated with a DNA vaccine expressing the tat, nef, gag, and env genes of the HIV-2UC2 group B isolate were partially protected against autologous challenge without the production of neutralizing antibodies (33). These studies illustrate the urgent need for new vaccine immunogens and/or vaccination strategies that elicit the production of broadly reactive NAbs against HIV-2. The present study was designed to investigate in the mouse model the immunogenicity and neutralizing response elicited by novel recombinant envelope proteins derived from the reference primary HIV-2ALI isolate, when administered alone or in different prime-boost combinations.     

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

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

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.Lentiviruses are a group of strictly exogenous retroviruses that infect a range of mammalian hosts. One characteristic of this group of retroviruses is the rapid sequence divergence observed between virus strains as well as different lentiviruses, which resulted in the evolution of viruses with large differences in genome organization and sequence (20). Most of the sequence homology between highly divergent lentiviruses is present in the gag and pol gene products (8, 21). Sequence homology between the envelope glycoproteins of different lentiviruses has previously been shown to occur only in the ectodomain of the transmembrane subunit (TM) but not in the surface unit (SU) glycoprotein (3, 8, 21–23). Due to this apparent lack of sequence conservation in lentiviral SU, it has been unclear how the SU of different lentiviruses are structurally related to each other. To address this question, we recently compared SU sequences from the gp120 from primate lentiviruses and the gp135 of small ruminant lentiviruses and found a statistically significant sequence similarity between the second conserved region (C2) of gp120 and a 99-amino-acid region from gp135 (10). Analysis of this gp120-gp135 sequence similarity in the context of the gp120 structure revealed a partial structural similarity between gp120 and gp135.The human immunodeficiency virus type 1 (HIV-1) gp120 core bound to CD4 is composed of two major domains, the inner and outer domains, and a minidomain composed of four antiparallel β-strands, the bridging sheet (13). Sequences from the C2 region form most of the β-strands of a two-helix, two-strand bundle and a five-stranded β-sandwich in the inner domain as well as some β-strands of the outer domain of gp120 (13). Most of the similarity motifs between gp135 and the C2 region of gp120 coincide with sequences corresponding to β-strands 4 through 8 in the HIV-1 gp120 inner domain and β-strands 11 and 12 in the outer domain (10). Significantly, all four cysteines that form two disulfide bonds in the proximal region of the gp120 inner domain as well as the first cysteine of the gp120 V3 loop in β12 (13, 15) are conserved in gp135, indicating a partial similarity between the tertiary structures of gp120 and gp135 (10).The most conserved sequences between gp120 and gp135 correspond to strands β4 and β5 in the five-stranded β-sandwich structure of the proximal region of the inner proximal domain of HIV-1 gp120. Two additional β-strands in this five-stranded β-sandwich are derived from C1 and C5 sequences of HIV-1 gp120 (13). We hypothesized that C1 and C5 sequences, which are part of a structurally conserved SU inner proximal domain, should also be conserved between gp120 and gp135 and possibly in the SU of other lentiviruses. Here we show that two short motifs located in the gp120 C2 and C5 regions which are part of an antiparallel β-sheet in the gp120 inner proximal domain are conserved in the lentiviruses, indicating that a surface of the inner domain of HIV-1 gp120 is conserved in the SU of other lentiviruses. In addition, the C2 motif is also present in the envelope glycoproteins encoded by A-type endogenous retroviral elements and type B retroviruses (type A and B retroviruses), suggesting a local structural similarity between the SU of lentiviruses and type A and B retroviruses.

Sequence motif of the C5 region of HIV-1 gp120 is present in the SU of all lentiviruses.

As the sequences of three of the five β-strands of the gp120 inner proximal domain β-sandwich are conserved in gp135, we first tried to determine whether the gp120-gp135 sequence similarity extends to the other two β-strands which are part of this structure. One of these strands is β1, located in the C1 region of gp120 (13). Although the sequence of β1 is relatively well conserved among the primate lentiviruses, it is only 3 amino acids long, and a reliable assignation of similar sequences in gp135 could not be done. The other strand of this β-sandwich structure is the 7-amino-acid long β25. This strand is antiparallel to β5, which is the most conserved sequence between gp120 and gp135 (10, 13). Strand β25 is located about 20 amino acid residues upstream from the carboxy terminus of HIV-1 gp120 in the C5 region, and its sequence is highly conserved among strains of primate lentiviruses (sequence KYKVVKI in HIV-1_HXB2; residues conserved between HIV-1 strains are underlined) (12, 24). The last residue of this motif has been shown to be important for anchoring of gp120 on gp41 (9), suggesting that β25 is a functionally important structure of the inner proximal domain of gp120 likely to be conserved in other lentiviral glycoproteins. Sequences similar to the HIV-1 gp120 β25 motif (C5 motif) were visually sought in the gp135 carboxy-terminal region. A similar sequence was found in the caprine arthritis-encephalitis virus (CAEV) and visna virus gp135 between 33 and 34 amino acid residues upstream from the carboxy terminus of gp135 (Fig. ​(Fig.1B).1B). Similar to the C5 motif sequence of primate lentiviruses, the gp135 C5 motif is highly conserved in the gp135 of small ruminant lentiviruses (4, 27, 31, 35, 36). The sequence similarity also included the strictly conserved residue L483 of HIV-1 gp120 in the preceding α-helix 5, which is part of the two-helix, two-strand bundle of the inner domain. Flanking regions of gp120 and gp135 did not show any sequence similarity (not shown). Due to its short length, the significance of the conservation of the C5 motif in gp120 and gp135 was unclear. If this motif is indeed part of a structurally or functionally important domain of SU and not due only to chance, it should also be conserved in the SU of other lentiviruses. Therefore, to establish the relevance of this sequence similarity, we determined whether the C5 motif was also present in the carboxy terminus of the SU of other lentiviruses. Open in a separate windowFIG. 1Alignment of the C2 (A) and C5 (B) motifs of the SU from lentiviruses and type A and B retroviruses. Numbers at the right of the alignments indicate the position of the last residue of the motif from the initiation codon. Letters above the alignment indicate residue positions within each motif. Black backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses for each position of the motifs. Gray backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses (but which are nonconservative with the residues in black background) for each position. Numbers in parentheses indicate the number of amino acids between the last position of the C5 motif and the carboxy terminus of SU for each lentivirus. Thick lines indicate sequences which are part of HIV-1 gp120 strands β4, β5, and β25 and helix α5 (13). HIV-1 and HIV-2, human immunodeficiency virus types 1 (strain HXB2, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382"}}K03455) and 2 (strain ROD, {"type":"entrez-nucleotide","attrs":{"text":"X05291","term_id":"60146"}}X05291); CAEV, caprine arthritis-encephalitis virus ({"type":"entrez-nucleotide","attrs":{"text":"M33677","term_id":"323294"}}M33677); Visna, visna virus ({"type":"entrez-nucleotide","attrs":{"text":"M10608","term_id":"470313"}}M10608); JSRV, jaagsiekte sheep retrovirus ({"type":"entrez-nucleotide","attrs":{"text":"M80216","term_id":"331338"}}M80216); EIAV, equine infectious anemia virus (AF033820); FIV, feline immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M73965","term_id":"323946"}}M73965); BIV, bovine immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M32690","term_id":"210706"}}M32690); JDV, jembrana disease lentivirus ({"type":"entrez-nucleotide","attrs":{"text":"U21603","term_id":"733067"}}U21603); HERV-K, human endogenous retrovirus K, type 2 genome ({"type":"entrez-nucleotide","attrs":{"text":"X82272","term_id":"757871"}}X82272); MMTV, mouse mammary tumor virus ({"type":"entrez-nucleotide","attrs":{"text":"X01811","term_id":"61630"}}X01811); MIAE, mouse intracisternal A-type element ({"type":"entrez-nucleotide","attrs":{"text":"M73818","term_id":"194061"}}M73818).Sequences conforming to the C5 motif consensus were also found in the SU of the equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and the bovine jembrana disease lentivirus (JDV), 19 to 23 amino acid residues from the carboxy terminus of SU, the same relative position as the C5 motif from the carboxy terminus of gp120 in primate lentiviruses (Fig. ​(Fig.1B).1B). This sequence similarity was clear when considering the chemical similarities of amino acid side chains (Gln/Glu, Tyr/Trp, Lys/Arg/Gln, Val/Leu, or Val/Ile). A survey of lentiviral SU sequences present in GenBank revealed that the C5 motif was also highly conserved between EIAV, FIV, and BIV strains. For example, the C5 motif was found to be strictly conserved in 64 of 69 EIAV gp90 sequences in GenBank and is also stable during in vivo persistent infection (16, 39). However, the little variation that is observed between strains of a given lentivirus follows the same pattern as variation between different lentiviruses, suggesting a common constraint on sequence variation in different lentiviruses. For example, position h of the C5 motif of HIV-1 gp120 can be either of the conservative variations Lys/Arg or Gln/Glu, the same amino acids present at position h in other lentiviruses. Similarly, position h of the C5 motif of CAEV in different strains is either Lys or Arg (35), two of the residues allowed at position h in HIV-1 gp120. In addition, position b in the C5 motif of most FIV gp100 sequences in GenBank is the conservative variation Gln or Glu, the same amino acids present at position b of the C5 motif in EIAV and the small ruminant lentiviruses, respectively. Although the C5 motif is present in all lentiviruses, the flanking sequences were not consistently conserved except for a few amino acids in some pairwise alignments (not shown). Therefore, although conservation of the C5 motif may not be statistically significant in some SU pairwise alignments, the presence of this motif in the same position relative to the carboxy terminus of SU in all lentiviruses indicates that strand β25 of gp120 is an important structural or functional domain conserved in all lentiviruses.

Sequences similar to an HIV-1 gp120 C2 motif are present in the SU of most lentiviruses.

Using computer-assisted searches, we were previously unable to find in EIAV, BIV, or FIV the same extensive region of similarity that is observed between the C2 region of gp120 and gp135 (10). However, the presence of the C5 (β25) motif in all lentiviruses suggests that sequences similar to gp120 β5, which is antiparallel to β25 and conserved between gp120 and gp135, are also present in degenerate form in other lentiviruses. Visual examination of SU sequences from different lentiviruses revealed the presence of a similar motif (C2 motif) in EIAV, BIV, and JDV although not in FIV (Fig. ​(Fig.1A).1A). This 12-amino-acid C2 motif encompasses most of gp120 β-strands 4 and 5 and includes a strictly conserved cysteine residue in the β4 region. The C2 motif is highly conserved between strains of EIAV and BIV. In EIAV, the C2 motif is stable during persistent infection, with few conservative changes observed (16, 39). In addition, the C2 motif was found to be strictly conserved in 176 of 179 EIAV gp90 sequences present in GenBank, despite considerable sequence variation in other regions.Although some positions of the C2 motif were not absolutely conserved, we found a common pattern of variation between distantly related lentiviruses. For example, position f of the C2 motif is either Pro in EIAV, HIV-2, and simian immunodeficiency virus or aromatic (Tyr or Trp) in the small ruminant lentiviruses BIV and JDV. Also, position h can be either Phe or Tyr even in closely related lentiviruses (HIV-1/HIV-2, visna virus/CAEV, or BIV/JDV), and position l can be either Arg or Lys in the primate and small ruminant lentiviruses or Gln, which is a common conservative substitution for Arg and Lys, in EIAV, BIV, and JDV. Therefore, the C2 motifs of different lentiviruses appear to have a common constraint on sequence variation, suggesting a structural or functional similarity between the HIV-1 gp120 C2 domain and the SU of EIAV, BIV, and JDV.The other previously described gp120-gp135 conserved motifs outside the β4/β5 region could not be identified in the SU of other lentiviruses, including the sequence of gp120 β8, which has a cysteine forming a disulfide bond with the conserved β4 cysteine. Although the C2 motif was not present in FIV gp100, a similar motif was identified in a location upstream from the FIV gp100 V3 region (sequence SYCTDPLQIPLI, amino acids 318 to 329; conserved residues are underlined), in a similar relative position from gp100 V3 as the C2 motif from the V3 region of HIV-1 gp120. However, some of the highly conserved positions of the motif (positions g, h, and j) were not conserved in FIV gp100, and the significance of this FIV gp100 motif is unclear.

C2 motif is present in type A and B retroviral envelope surface glycoproteins.

The conservation of two short motifs in distant regions of SU that are located close to each other in the tertiary structure of HIV-1 gp120 suggests that this region represents a domain of SU that is of structural or functional importance. The TM ectodomains from lentiviruses and type B retroviruses have been shown to have some sequence similarity (19, 34, 38). Therefore, we asked whether sequence similarity between the Env of lentiviruses and type B retroviruses extends to the C2 and C5 motifs of SU.The type A and B retroviruses have some sequence homology in SU, and most of the sequence homology is located in the carboxy-terminal region of SU (18, 38). Visual examination of SU sequences from the human endogenous retrovirus K (18), mouse intracisternal A-type element (26), the exogenous/endogenous mouse mammary tumor virus (25), and the exogenous/endogenous type B/D jaagsiekte sheep retrovirus (JSRV) and the closely related ovine enzootic nasal tumor virus (which encode type B retroviral envelopes) (6, 38) revealed a sequence closely related to the C2 motif in their conserved carboxy-terminal region (Fig. ​(Fig.1A).1A). This sequence represents one of the most conserved sequences in the SU of this group of retroviruses and is also conserved among different strains or members of endogenous families (not shown). Some positions of the C2 motif, such as positions c, d, and g, are strictly or almost completely conserved between the lentiviruses and type A and B retroviruses. However, more informative than the sequence similarity between lentiviruses and type A and B retroviruses is the lack of distinction between the patterns of sequence variation for each position of the motif within and between retrovirus groups, even between closely related viruses. For example, position e of the C2 motif within both the lentiviruses and type A and B retroviruses can be either Pro or basic/Gln; the “dimorphic” position f encodes only Tyr/Trp or Pro (except in HIV-1); position h encodes either Phe or Tyr in all sequences; position i encodes either Ala or a hydrophobic residue in most sequences; position j encodes either Ile, Leu, or Phe in all sequences; position k encodes either Leu, Ile, or Val in all sequences; and position l is preferentially Lys, Arg, or Gln in the lentiviruses and JSRV. Most of these degenerate positions represent very conservative variations (positions a and h through l) or a restricted number of nonconservative variations (positions e and f, in the turn between β4 and β5). The sequence conservation and common pattern of variation between the C2 motifs of lentiviruses and type A and B retroviruses indicate a similar structural or functional constraint on sequence variation in the SU of these two groups of viruses.In contrast to the type A and B retroviruses, sequences similar to the C2 or C5 motifs could not be found in the SU of the Moloney murine leukemia virus, bovine leukemia virus, human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), Rous sarcoma virus, feline RD114 endogenous retrovirus, baboon endogenous retrovirus, feline leukemia virus type A, the Mason-Pfizer monkey retrovirus, or any spumaretrovirus even when using the Findpatterns program of the GCG package (7).Here we show that two short SU motifs are highly conserved in the lentiviruses and that one of these motifs is also conserved in the type A and B retroviruses. Many of the pairwise alignments were not statistically significant when tested by the Monte Carlo simulation of the Bestfit program of the GCG package and could therefore be attributed to chance. However, when all lentiviral sequences are included in the analysis and the multiple alignment is interpreted in the context of the X-ray structure of HIV-1 gp120, the conserved C2 and C5 motifs have a clear structural significance. The conservation of these motifs indicates that the region of the HIV-1 gp120 inner proximal domain centered on the antiparallel β-strands 5 and 25 forms a highly conserved lentiviral SU surface and suggests a possible structural similarity between the SU of lentiviruses and type A and B retroviruses in that domain. Although the C2 motif is too short to rule out convergent evolution between the SU of lentiviruses and type A and B retroviruses, their sequence similarity in TM (19, 34, 38) supports a common origin for most or the entire env genes of these two retroviral groups.The reason for the disagreement between the different degrees of sequence similarity in the SU of lentiviruses and the phylogenetic analyses of the pol gene products is unclear but probably reflects differences in evolutionary rates in different lentiviruses or recombination events (19, 20). Precedents for recombination events between env genes of closely or distantly related retroviruses, deduced from phylogenetic analyses, have been described. An exchange of env sequences probably occurred between HTLV-1 and HTLV-2 (19) and between a type C retrovirus closely related to the avian reticuloendotheliosis virus and a type B retrovirus which originated the type D retroviruses (19, 38).Modeling of the trimeric SU complex on the virion surface indicates that strands β5 and β25 form part of the most virion-proximal surface of the gp120 core (14, 37). While none of the residues of the C2 motif was directly tested for interactions with TM, at least one of the residues of β25 in the C5 region of HIV-1 gp120, I491, is important for stable SU-TM interactions (9). Therefore, the conserved lentiviral SU surface may represent a common structure among lentiviruses and possibly type A and B retroviruses for anchoring SU on TM in the envelope glycoprotein complex. It is interesting that the C5 motif region, which forms a β-strand in the CD4-bound gp120 core, is included in a computer-modeled pocket structure postulated to be important in SU-TM interactions (28), suggesting a structural basis for SU shedding upon receptor-induced conformational change.The sequence of the HIV-1 gp120 outer domain, shown as a cross-hatched box in Fig. ​Fig.2,2, is included entirely between the C2 and C5 motifs (13). Our previous sequence analysis indicates that the gp135s of small ruminant lentiviruses have a similar inner/outer domain organization: most strands of the inner domain β-sandwich as well as β12, located in the outer domain immediately upstream from the gp120 V3 loop, are conserved between gp135 and the gp120 of primate lentiviruses (10). The identification of a homologue of gp120 β25 in gp135 about 290 amino acid residues downstream from the C2 motif provides further support for a similar domain organization in the SU of primate and small ruminant lentiviruses. Consistent with this interpretation, the putative outer domain of gp135, located between the C2 and C5 motifs, is highly glycosylated and contains more than 80% of the potential N-linked glycosylation sites of gp135 (11), similar to the heavy glycosylation of the gp120 outer domain (37). In this gp135 domain model, the distance between the C2 and C5 motifs in the primary structure of SU would indicate a larger relative size of the putative outer domain of gp135 than gp120 outer domain. The presence of the C2 and C5 motifs in EIAV, BIV, and JDV would also suggest an analogous inner/outer domain organization for the SU of these lentiviruses. However, the shorter sequence between the C2 and C5 motifs in EIAV, BIV, and JDV may indicate either a much smaller or absent outer domain in the SU of these viruses (Fig. ​(Fig.2).2). The conserved C2 motif of EIAV gp90 was shown to be part of a minor neutralization epitope recognized by a murine monoclonal antibody (1), suggesting that the EIAV C2 motif is better exposed on the virion surface than the C2 motif of gp120, compatible with a smaller or absent outer domain in gp90. Interestingly, the C2 motif of type A and B retroviruses is located in the carboxy terminus of SU (Fig. ​(Fig.2)2) and C5 appears to be absent, indicating that the surface glycoproteins of type A and B retroviruses, although possibly structurally related to the SU of lentiviruses, probably lack an outer domain homologue and have a different domain organization than the SU of lentiviruses. Open in a separate windowFIG. 2Location of the C2 and C5 motifs in retrovirus envelope glycoproteins. The Env glycoproteins (excluding the amino-terminal leader peptide) are drawn to scale and aligned by the SU-TM cleavage sites conserved in all retroviruses (dotted line). The SU and TM domains of Env are indicated by double arrows. The boundaries of the C2, V3, and C5 regions of HIV-1 gp120 are indicated by thick lines above the alignment, and the location of the HIV-1 gp120 outer domain sequence is shown by a cross-hatched box. The black and gray boxes in the SU domain indicate the positions of the C2 and C5 motifs, respectively. Asterisks represent the described PNDs of EIAV (1), visna virus (29), FIV (17), and T-cell-adapted strains of HIV-1 (33) and HIV-2 (2).The two conserved colinear motifs of lentivirus SU could be useful as structural points of reference for comparative structural studies of SU from different lentiviruses. Variable domains of SU are important in the mechanisms of host cell invasion, tropism determination, and immune evasion. In HIV-1, the third variable loop V3 of gp120 is the main target of neutralizing antibodies in tissue culture-adapted strains and also determines coreceptor usage and tropism (5, 30, 32, 33). Whether sequences in variable regions of SU in other lentiviruses that are functionally equivalent to the gp120 V3 loop are also structurally related to the gp120 V3 loop is not clear. The position of variable domains relative to the C2 and C5 motifs could therefore indicate their structural relationship. For example, the principal neutralization domain (PND) of EIAV gp90, postulated to be functionally equivalent to the gp120 V3 loop (1, 16), is located upstream from the C2 motif instead of downstream, as the V3 loop in gp120 is (Fig. ​(Fig.2),2), suggesting that the gp90 PND and the gp120 V3 loop, while having similar roles in evasion of humoral immune responses, may not be structurally related to each other. A similar situation also occurs in visna virus, whose PND, located in the carboxy-terminal region of gp135 (29), was previously shown to be structurally unrelated to the HIV-1 gp120 V3 loop (10). This would indicate that different lentiviruses may have evolved different regions of a primordial lentivirus surface glycoprotein to perform similar functions important in virus-host interactions.     

Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect

The Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.Entry of enveloped viruses into mammalian cells requires that the virion envelope fuse with the cell membrane after attachment to the cell surface. Herpesviruses require the functions of multiple protein species to mediate this event, and in keeping with the common origin and diverse habitats of these viruses, some of the proteins involved in penetration appear to be conserved throughout the family and some appear to be restricted to individual members or more closely related members with similar tropism. The two glycoproteins gH and gL fall into the first category of conserved proteins. Glycoprotein gH has been implicated as a major player in virus-cell fusion in many herpesviruses (8, 10, 11, 22, 28, 32, 34), and gL is an essential partner which is required for folding and transport of gH out of the endoplasmic reticulum (6, 19, 21, 27, 28, 35, 38, 45). The gH and gL homologs of Epstein-Barr virus (EBV) are gp85, the product of the BXLF2 open reading frame (ORF) (13, 31), and gp25, the product of the BKRF2 ORF (45), and these homologs appear to behave much as their counterparts in other herpesviruses do (45). However, a third glycoprotein, gp42, associates with the EBV gH-gL complex and falls into the second category of proteins, those with a more restricted distribution.Glycoprotein gp42 is the product of the BZLF2 ORF (26), and although there may be a functionally similar protein in cytomegalovirus (18, 24), it is not predicted to have a homolog in other human herpesviruses. It does, however, have a homolog in ORF51 of equine herpes virus 2 (43). Both EBV and equine herpes virus 2 infect B lymphocytes (1), and several lines of evidence suggest that, at least in the case of EBV, gp42 is critical to the infection of this cell type. A monoclonal antibody (MAb) called F-2-1 that reacts with gp42 has no affect on EBV attachment to its receptor, complement receptor type 2 (CR2) (CD21), but inhibits fusion of the virus with the B-cell membrane and neutralizes infection (29). Glycoprotein gp42 interacts with the β₁ domain of the HLA class II protein HLA-DR (39), and MAb F-2-1 interferes with this interaction (25). Like F-2-1, a MAb to HLA-DR or a soluble form of gp42 can block B-cell transformation, and B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of HLA class II is restored (25). Collectively these observations suggest that gp42, probably by virtue of its interaction with HLA class II, is essential to infection of the B lymphocyte. To answer directly the question of whether gp42 is an indispensable glycoprotein, we derived a virus that could be definitively shown to lack expression of the molecule and examined its ability to infect normal resting B lymphocytes. We report here that virus with expression of gp42 blocked can exit cells normally and can bind to receptor-positive target cells. However, it is unable to penetrate into cells and initiate infection.     

Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).Human T-cell lymphotropic virus type 1 (HTLV-1) is a type C retrovirus and the etiologic agent of adult T-cell leukemia (43, 56, 59) and HTLV-1-associated myelopathy or tropical spastic paraparesis (15, 17, 49, 61). Although HTLV-1 shows tropism primarily for T cells, it can infect a variety of cell types including cells from some nonhuman species (6, 9, 27, 46, 48, 60, 62). Infection by free HTLV-1 tends to be highly inefficient, and the virus appears to be transmitted primarily by the cell-to-cell route (37). The HTLV-1 envelope glycoprotein is synthesized as a 61-kDa precursor which is cleaved into surface (gp46) and transmembrane (gp21) proteins (40, 57). gp46 is thought to serve as the virus attachment protein, as does gp120 for human immunodeficiency virus (HIV) (40, 57). Although previous reports have identified host cell molecules which might potentially mediate virus binding (9, 14), the cellular receptor for HTLV-1 has not been definitively identified. A recent study in which affinity chromatography was carried out with a gp46 peptide has provided evidence that the heat shock protein HSC70 binds directly to gp46 and may serve as a virus receptor (47).gp21 contains an N-terminal hydrophobic fusion domain and likely serves as a fusion protein similar to HIV gp41 (12, 61). Like many other retroviruses, HTLV-1 can induce syncytium formation between infected cells and certain uninfected cell types (28, 39). However, there are no data to indicate that virus transmission or virus persistence in vivo depends on syncytium formation. It is thought that cell-cell fusion involves the same receptors and occurs in a manner similar to virus-cell fusion. For this reason, HTLV-1 syncytium assays have been used to screen for cell surface molecules that may serve as virus receptors (13, 14, 25, 29). Monoclonal antibodies (MAbs) against a number of membrane proteins including members of the tetraspanner family (30, 31) have been found to block syncytium formation. My colleagues and I recently reported that expression of the cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) on uninfected cells can confer sensitivity to HTLV-1-mediated syncytium formation (25). In this previous study, we were not able to block HTLV-1 cell fusion with MAbs against the major VCAM-1 counterreceptor VLA-4 (25). Others have reported that MAbs to other adhesion molecules including intercellular adhesion molecule 3 (ICAM-3) also block HTLV-1 syncytium formation (29). We have demonstrated that adhesion molecules also facilitate HIV type 1 (HIV-1) infection and syncytium formation (16, 24). Thus, adhesion molecules may be important accessory molecules for retroviruses generally.Earlier studies on accessory molecules involved in HTLV-1 biology have been extended by immunizing mice with HTLV-1-infected cells and screening for MAbs that block VCAM-1-supported HTLV-1 syncytium formation. Four new MAbs that completely block HTLV-1-mediated cell fusion have been generated. The MAbs were all determined to be specific for class II major histocompatibility complex (MHC) molecules. These MAbs had no effect on syncytium formation induced by HIV-1. Studies on the mechanism by which the MAbs mediate this effect have revealed a novel mode of antibody blockade of virus-induced cell fusion: protein crowding at the infected cell surface resulting in steric blockade of critical receptor-ligand interactions.     

Extent of Antigenic Diversity in the V3 Region of the Surface Glycoprotein,gp120, of Human Immunodeficiency Virus Type 1 Group M and Consequences for Serotyping

Human immunodeficiency virus type 1 (HIV-1) may be studied by molecular or immunological approaches. Most analyses have been performed by genetic comparison of isolates and have led to the definition of clades or subtypes within the major (M) group of HIV-1. Five subtypes (A to E) were initially identified by comparison of genomic sequences. Four new subtypes (F to I) were identified more recently. Amino acid differences in the immunogenic V3 loop between isolates have also been studied, leading to a phenetic classification of at least 14 clusters (1 to 14) of sequences (B. T. M. Korber, K. McInnes, R. F. Smith, and G. Myers, J. Virol. 68:6730–6744, 1994). In this study, we compared the antigenicity of the V3 consensus sequences defined by phylogenetic analysis to the antigenicity of those defined by phenetic analysis. We used a recently developed subtype-specific enzyme immunoassay (SSEIA) that uses the principle of blocking with an excess of peptide in the liquid phase. Two SSEIAs were performed, the first with five V3 sequences defined by phylogenetic analysis and the second with 14 V3 sequences defined by phenetic analysis. A total of 168 HIV-1 sera taken from seropositive individuals from seven different countries or regions were studied. Experimental and statistical data, including correlation matrix and cluster analyses, demonstrated associations between the genetic subtypes and phenetically associated groups. Most of these were predicted by Korber et al. (J. Virol. 68:6730–6744, 1994) by theoretical analysis. We also found that V3 sequences can be grouped into between three and five antigenically unrelated categories. Residues that may be responsible for major antigenic differences were identified at the apex of the V3 loop, within the octapeptide xIGPGxxx, where x represents the critical positions. Our study provides evidence that there is a limited number of V3 serotypes which could be easily monitored by serological assays to study the diversity and dynamics of HIV-1 strains.The diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem in the development of an effective vaccine against AIDS. Many HIV-1 sequences are now available, and phylogenetic analysis resulting in a continuously developing classification into subtypes or clades is possible (45). HIV-1 isolates are classified into the M group (for major) or O group (for outlier). The O group contains only a few variants, all from a limited area of Africa (19, 27, 50). The M group includes variants responsible for the present AIDS pandemic. It contains at least five subtypes (A to E), to which have been added more recently four other subtypes (F to I) (23, 28, 34, 36, 37). Subtypes A, C, D, G, and H are common in Africa (21, 35, 37, 38). Subtype B is the most common in America and Europe (24, 26, 51). Subtype E occurs mainly in Asia (25, 30, 41), and subtype F has been detected in Brazil and Romania (3, 28, 34). These distributions are not restrictive. Subtype C is also present in Asia (India and China), and subtype G is also present in Russia (7, 12, 29). The African subtypes (A, C, and D) and the Asian subtype (E) have also been identified in North America and in European countries (9, 13, 14, 32, 48). All the subtypes are present in Africa, including B (detected in West Africa), E (Central African Republic), and F (Cameroon) (1, 35, 38). Analysis of the genetic diversity of HIV-1 is becoming more difficult due to the increasing frequency of coinfections and recombinations (15, 20, 44).Phylogenetic trees have been generated with gag, env, or tat nucleotide sequences. Shorter DNA sequences encoding the functionally important V3 region of the envelope protein are most frequently used to provide reliable subtype designations (37). The diversity of the immunogenic V3 loop has also been studied by comparing the amino acids of different isolates, leading to a phenetic classification of at least 14 clusters of sequences, each one characterized by a consensus sequence based on the most common amino acid in a given position (22).The heterogeneity of HIV-1 strains is studied mostly by molecular characterization of genomic sequences. This involves sequencing fragments amplified by the PCR or the use of the heteroduplex mobility assay (10, 11). However, although these methods allow direct subtype classification, they are time-consuming and expensive and require highly trained workers. Serotyping of HIV-1 by antibody (Ab) binding to the V3 region has been suggested as an alternative approach (8, 40, 49, 51). Such an approach may make it possible to identify subtypes based on antigenic rather than genetic properties. This immunological information about antigenic diversity might be of value in vaccine development. We recently developed a subtype-specific enzyme immunoassay (SSEIA) which gave results consistent with those of genotyping (4, 48). This assay used V3 consensus sequences defined by genetic classification, so we wanted to compare the antigenicity of these V3 consensus sequences to the antigenicity of those defined by phenetic analysis. The phenetic clustering of V3 loop amino acid sequences is not always consistent with phylogenetic analysis. Our results suggested that a limited number of serotypes may exist and identified amino acids at the tip of the V3 loop that may be responsible for serological discrimination.