A Maraviroc-Resistant HIV-1 with Narrow Cross-Resistance to Other CCR5 Antagonists Depends on both N-Terminal and Extracellular Loop Domains of Drug-Bound CCR5

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.Small-molecule CCR5 antagonists are a relatively new class of drugs that block HIV entry into target cells, with the first member of this class, maraviroc (MVC), having been approved for the treatment of HIV-infected patients. These drugs bind to a hydrophobic pocket formed by the transmembrane helices of CCR5, inducing conformational changes in the extracellular loops (ECLs) of the receptor (18, 31, 39, 40, 58, 62, 64). These conformational changes can vary with different drugs, as evidenced by differential chemokine binding and HIV resistance profiles, and block the ability of HIV to use drug-bound CCR5 as a coreceptor for entry (59, 64).As with other antiretroviral agents, HIV can develop resistance to CCR5 antagonists. One pathway by which HIV can become resistant to CCR5 antagonists is via mutations in the viral envelope (Env) protein that enable it to recognize the drug-bound conformation of the coreceptor. Most of our information on this pathway has come from in vitro passaging of HIV-1 in the presence of increasing concentrations of inhibitor (2, 4, 5, 33, 41, 44, 61, 66). In most instances, the viral determinants of resistance are localized to the V3 loop of gp120 (5, 33, 41, 44, 46, 63, 66). This is as expected: the base of the V3 loop interacts with O-sulfated tyrosines in the N terminus of CCR5, while the tip of the V3 loop is thought to contact the ECLs of the receptor (14, 15, 17, 19, 26, 29, 37). Viral resistance to one CCR5 antagonist commonly results in cross-resistance to other drugs in this class, although this is not universally the case (33, 41, 60, 63, 66). Mechanistically, a number of CCR5 antagonist-resistant viruses have been shown to have increased dependence on the N-terminal domain of CCR5 (5, 34, 44, 45, 48), which is largely unaffected by drug binding and may allow viruses to tolerate drug-induced changes in ECL conformation.In contrast to several well-characterized viruses that have evolved resistance to CCR5 antagonists in vitro, few examples of patient-derived CCR5 antagonist-resistant viruses have been reported. One mechanism of resistance that has been described in patients is the outgrowth of CXCR4-tropic HIV isolates that were present at low frequencies prior to the initiation of therapy (22, 23, 35, 36, 42, 65). Due to this finding, patients undergo tropism testing prior to treatment with CCR5 antagonists, with only those harboring exclusively R5-tropic viruses considered candidates for therapy. Patient-derived viruses capable of using drug-bound CCR5 have been reported in studies using vicriviroc and aplaviroc (45, 60, 63). The aplaviroc-resistant viruses were determined to utilize the drug-bound form of the receptor by interacting primarily with the N terminus of CCR5, similar to the viruses derived by serial in vitro passaging (48).In the present study, we report the isolation of MVC-resistant Envs from a treatment-experienced patient who had a viral load rebound while on a regimen containing MVC. Viral Envs isolated from this patient at the time MVC therapy was initiated were fully sensitive to drug. However, resistance evolved over the course of 224 days, culminating in Envs that were completely resistant to inhibition but continued to use CCR5 for entry. The emergence of resistance was dependent upon changes within the V3 loop of the virus, while changes in the V4 loop modulated the magnitude of resistance. The MVC-resistant Envs studied here exhibited several unusual properties. First, while they were cross-resistant to TAK779, they remained sensitive to all other CCR5 antagonists tested, including vicriviroc and aplaviroc. Second, the Envs were particularly adept at utilizing low levels of CCR5 to mediate infection of cells. Third, and in contrast to several recent reports of CCR5 antagonist-resistant viruses, these Envs were dependent upon residues within both the N terminus and ECLs of CCR5 for efficient entry in the presence of drug. When considered in the context of other reports, our data suggest a model in which resistance to multiple CCR5 antagonists can arise if an Env protein becomes highly dependent upon the N-terminal domain of CCR5, the conformation of which appears to be unaffected by drug binding. A more narrow resistance profile results from changes in Env that enable it to use both the N-terminal domain of CCR5 as well as the drug-induced conformation of the CCR5 ECLs.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.     

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.     

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.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

Envelope Vaccination Shapes Viral Envelope Evolution following Simian Immunodeficiency Virus Infection in Rhesus Monkeys

The evolution of envelope mutations by replicating primate immunodeficiency viruses allows these viruses to escape from the immune pressure mediated by neutralizing antibodies. Vaccine-induced anti-envelope antibody responses may accelerate and/or alter the specificity of the antibodies, thus shaping the evolution of envelope mutations in the replicating virus. To explore this possibility, we studied the neutralizing antibody response and the envelope sequences in rhesus monkeys vaccinated with either gag-pol-nef immunogens or gag-pol-nef immunogens in combination with env and then infected with simian immunodeficiency virus (SIV). Using a pseudovirion neutralization assay, we demonstrate that envelope vaccination primed for an accelerated neutralizing antibody response following virus challenge. To monitor viral envelope evolution in these two cohorts of monkeys, full-length envelopes from plasma virus isolated at weeks 37 and 62 postchallenge were sequenced by single genome amplification to identify sites of envelope mutations. We show that env vaccination was associated with a change in the pattern of envelope mutations. Prevalent mutations in sequences from gag-pol-nef vaccinees included deletions in both variable regions 1 and 4 (V1 and V4), whereas deletions in the env vaccinees occurred only in V1. These data show that env vaccination altered the focus of the antibody-mediated selection pressure on the evolution of envelope following SIV challenge.Immune containment of human immunodeficiency virus (HIV-1) is complicated by the continuous genetic evolution of the virus. The evolution of the HIV-1 envelope is shaped, in part, by selective pressure of neutralizing antibodies (6, 12, 27, 34-36, 40). Changes in envelope sequence and glycosylation patterns following infection can allow the virus to escape neutralization. If the rate and extent of envelope sequence evolution following infection can be decreased, immune containment of HIV-1 may be improved.One possible strategy for modifying envelope evolution is vaccination prior to infection. A vaccine-elicited memory immune response could focus and potentiate the humoral immune response that develops following infection. The possible consequence of vaccination has not been assessed, however, because of the limited number of human volunteers who have received highly immunogenic envelope immunogens and subsequently became infected with HIV-1.Simian immunodeficiency virus (SIV) infection of rhesus monkeys provides a powerful model to study the effect of vaccination on envelope evolution. Like HIV-1, SIV employs both the CD4 molecule and the chemokine receptor CCR5 to enter a target cell and cause an AIDS-like disease in macaques (16, 22). Both SIV and HIV-1 envelopes are heavily glycosylated, with approximately 50% of their mass derived from carbohydrates (14, 21). SIV and HIV-1 envelopes share approximately 40% amino acid homology (10, 11) and have overlapping variable and constant regions, although the variable region 3 (V3) of HIV-1 envelope does not align with the homologous region of SIV envelope (7). Following SIV infection in rhesus monkeys, SIV envelope evolves most rapidly in variable regions 1 and 4 (V1 and V4, respectively), leading to nucleotide additions, deletions, and/or mutations that can potentially translate to changes in glycosylation (7, 9, 13, 15, 19, 29, 30).Studies done to characterize SIV neutralization suggest that it occurs through mechanisms similar to those seen in HIV-1 neutralization. Amino acid mutations in the envelope of both viruses contribute to the evasion of antibody binding directly by changing recognition sequences and/or envelope conformation. In addition, the glycosylation of envelope serves as a further obstacle to antibody recognition (20, 33, 40). Considerable effort has been devoted to defining neutralizing epitopes of the HIV and SIV envelopes. The known neutralizing human monoclonal antibodies elicited during natural infection are directed against HIV-1 envelope target sites on both gp120 and gp41, including the V3 region, the CD4 binding site, oligomannose residues of gp120, and gp41 (17, 31). The neutralizing epitope profile of SIV envelope includes the CD4 binding site and gp41 but not the V3 region. There is conflicting evidence as to whether V1, V2, and/or V4 of SIV are targets for antibody neutralization (15, 18, 19). The present study addresses whether vaccine-induced immune responses accelerate the generation of autologous neutralizing antibodies following SIV challenge in rhesus monkeys and how this humoral immune response can potentially shape viral sequence evolution.     

Different Tempo and Anatomic Location of Dual-Tropic and X4 Virus Emergence in a Model of R5 Simian-Human Immunodeficiency Virus Infection

We previously reported coreceptor switch in rhesus macaques inoculated intravenously with R5 simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N). Whether R5-to-X4 virus evolution occurs in mucosally infected animals and in which anatomic site the switch occurs, however, were not addressed. We herein report a change in coreceptor preference in macaques infected intrarectally with SHIV_SF162P3N. The switch occurred in infected animals with high levels of virus replication and undetectable antiviral antibody response and required sequence changes in the V3 loop of the gp120 envelope protein. X4 virus emergence was associated with an accelerated drop in peripheral CD4⁺ T-cell count but followed rather than preceded the onset of CD4⁺ T-cell loss. The conditions, genotypic requirements, and patterns of coreceptor switch in intrarectally infected animals were thus remarkably consistent with those found in macaques infected intravenously. They also overlapped with those reported for humans, suggestive of a common mechanism for coreceptor switch in the two hosts. Furthermore, two independent R5-to-X4 evolutionary pathways were identified in one infected animal, giving rise to dual-tropic and X4 viruses which differed in switch kinetics and tissue localization. The dual-tropic switch event predominated early, and the virus established infection in multiple tissues sites. In contrast, the switch to X4 virus occurred later, initiating and expanding mainly in peripheral lymph nodes. These findings help define R5 SHIV_SF162P3N infection of rhesus macaques as a model to study the mechanistic basis, dynamics, and sites of HIV-1 coreceptor switch.The human immunodeficiency virus (HIV) enters target cells via binding of the viral envelope glycoprotein to the CD4 receptor, triggering envelope conformational changes that allow for interaction with either the CCR5 or CXCR4 chemokine receptor (1, 3, 8, 15, 16, 18). Most HIV type 1 (HIV-1) transmissions are initiated with CCR5-using (R5) viruses (58, 68). With time, CXCR4-tropic (X4) viruses emerge and coexist with R5 viruses in close to 50% of subtype B-infected individuals, and this is accompanied by a rise in viremia, rapid CD4⁺ T-cell loss, and progression to disease (4, 7, 11, 34, 57, 65). The mechanistic basis and reasons for HIV-1 coreceptor switch, however, are still not well understood. Several factors including high viral load, low CD4⁺ T-cell numbers, reduced availability of CCR5⁺ cells, and progressive immune dysfunction have been proposed as playing important roles (48, 54). Since X4 virus emergence is associated with a faster rate of disease progression, insights into the determinants of HIV-1 coreceptor switch are of interest in understanding viral pathogenesis. Furthermore, with the introduction of CCR5 entry inhibitors as anti-HIV therapeutics (19, 23, 24, 38), there is a need not only to identify the presence of X4 variants in patients when treatment options are considered but also to understand the factors that influence X4 virus evolution. Although the majority of individuals failing on short-term CCR5 antagonist monotherapy harbor preexisting minor X4 variants (71), it is conceivable that given the right conditions and selective forces, inhibiting HIV-1 entry via CCR5 may drive the virus to evolve to CXCR4 usage and exacerbate disease. An animal model that faithfully recapitulates the process of coreceptor switch will be highly useful to study and identify the determinants and conditions that facilitate the change in coreceptor preference. In addition, an animal model provides the opportunity to track the kinetics of coreceptor switching at different anatomical sites, which may inform on the mechanisms of X4 virus emergence.In this regard, we recently reported coreceptor switch in two of nine rhesus macaques (RM) inoculated intravenously with simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N) that bears an HIV-1 CCR5-tropic Env (28, 29). In order to establish a reproducible model for coreceptor switch, however, it was crucial to document additional switching events. Furthermore, since the majority of HIV transmission occurs via mucosal surfaces, it was important to demonstrate coreceptor switch in macaques infected with R5 SHIV_SF162P3N by the mucosal route to validate this animal model in studying the in vivo evolution of HIV-1 coreceptor usage. Additionally, the tissue compartment(s) where CXCR4-using viruses evolve and expand is not well characterized. A recent study indicates that the thymus may play an important role in the evolution and/or amplification of coreceptor variants in pediatric HIV infection (56). Since the thymus is the primary source of T lymphopoiesis during early life (45) and since CXCR4 is the predominant coreceptor expressed on thymocytes (33, 64), this organ would seem to provide the ideal milieu for X4 amplification in infants and children. Indeed, we previously showed that whereas X4 SHIV infection of newborn RM resulted in severe thymic involution, R5 SHIV infection induced only a minor disruption in thymic morphology (55), lending support to the idea that the thymus is a preferred site for X4 replication in pediatric HIV infections. Nevertheless, thymopoietic function declines with age (17, 42, 60), and naïve T cells that express high levels of CXCR4 are also enriched in peripheral lymph nodes (5, 27, 36, 66). Thus, the role of the thymus and other lymphoid tissues in HIV-1 coreceptor switch in older individuals remains to be determined. To address these issues, we inoculated adult RM intrarectally (i.r.) with R5 SHIV_SF162P3N and performed frequent longitudinal blood and tissue samplings. Our goal was to document changes in coreceptor preference in mucosally infected macaques, as well as to obtain a more detailed picture of the kinetics and site of X4 virus evolution and amplification in vivo.     

Vaccine protection by live, attenuated simian immunodeficiency virus in the absence of high-titer antibody responses and high-frequency cellular immune responses measurable in the periphery

An attenuated derivative of simian immunodeficiency virus strain 239 deleted of V1-V2 sequences in the envelope gene (SIV239ΔV1-V2) was used for vaccine/challenge experiments in rhesus monkeys. Peak levels of viral RNA in plasma of 10⁴ to 10^6.5 copies/ml in the weeks immediately following inoculation of SIV239ΔV1-V2 were 10- to 1,000-fold lower than those observed with parental SIV239 (∼10^7.3 copies/ml). Viral loads consistently remained below 200 copies/ml after 8 weeks of infection by the attenuated SIV239ΔV1-V2 strain. Viral localization experiments revealed large numbers of infected cells within organized lymphoid nodules of the colonic gut-associated lymphoid tissue at 14 days; double-labeling experiments indicated that 93.5% of the virally infected cells at this site were positive for the macrophage marker CD68. Cellular and humoral immune responses measured principally by gamma interferon enzyme-linked immunospot and neutralization assays were variable in the five vaccinated monkeys. One monkey had responses in these assays comparable to or only slightly less than those observed in monkeys infected with parental, wild-type SIV239. Four of the vaccinated monkeys, however, had low, marginal, or undetectable responses in these same assays. These five vaccinated monkeys and three naïve control monkeys were subsequently challenged intravenously with wild-type SIV239. Three of the five vaccinated monkeys, including the one with strong anti-SIV immune responses, were strongly protected against the challenge on the basis of viral load measurements. Surprisingly, two of the vaccinated monkeys were strongly protected against SIV239 challenge despite the presence of cellular anti-SIV responses of low-frequency and low-titer anti-SIV antibody responses. These results indicate that high-titer anti-SIV antibody responses and high-frequency anti-SIV cellular immune responses measurable by standard assays from the peripheral blood are not needed to achieve strong vaccine protection, even against a difficult, neutralization-resistant strain such as SIV239.The characteristics of human immunodeficiency virus type 1 (HIV-1) infection suggest major difficulty for the development of a preventive vaccine (19, 23). Pessimism regarding the prospects for a vaccine is derived at least in part from the ability of HIV-1 to continually replicate in the face of apparently strong host immune responses, resistance to antibody-mediated neutralization, and the extensive sequence diversity in field strains of the virus. Lack of knowledge regarding the key components of a protective immune response also remains a major scientific obstacle. Vaccine/challenge experiments with macaque monkeys have been used to evaluate the properties and relative effectiveness of different vaccine approaches and to gauge the formidable nature of these difficulties.One lesson that has been learned from vaccine/challenge experiments with macaque monkeys is the importance of challenge strain on outcome. Vaccinated monkeys that have been challenged with strains of simian immunodeficiency virus (SIV) with an HIV-1 envelope (SHIV) have almost invariably exhibited strong, long-term protection against disease, irrespective of the nature of the vaccine. Even peptide immunogens have protected against SHIV-induced disease (6, 12, 38). Vaccine approaches that have protected against SHIV challenge include DNA (5, 13), recombinant poxvirus (4), recombinant adenovirus (57), other viral recombinants (18, 55), prime and boost protocols (3, 53, 65), and purified protein (10, 64). Vaccine protection against pathogenic SIV strains such as SIV239, SIV251, and SIV-E660 has been much more difficult to achieve (2, 11, 27, 63). The identical replication-defective gag-recombinant adenovirus that provided strong protection against SHIV challenge (57) provided little or no protection against SIV239 challenge (11). Disappointing levels of protection against SIV have often been observed in the face of apparently robust vaccine-induced immune responses (see, for example, Vogel et al. [63] and Casimiro et al. [11]). Some partial vaccine protections against these SIV strains have been achieved by recombinant poxvirus (7, 50), replication-competent recombinant adenovirus (51), replication-defective adenovirus (66), recombinant poliovirus (15), recombinant Venezuelan equine encephalitis virus (18), and recombinant Sendai virus (44).Differences between the biological properties of the SIV strains and those of the SHIV strains used for the above-mentioned studies provide clues as to what may be responsible for the differences in outcome. These SIV strains are difficult to neutralize (26, 34), use CCR5 as a coreceptor for entry into cells (21, 52), and induce a chronic, progressive disease course (17), and this course is independent of the infectious dose (17). The SHIV strains used for the above-mentioned studies are easier to neutralize, use CXCR4 for entry, and induce an acute decline in CD4 counts, and the disease course is dose dependent (29, 30, 48, 54). These SIV strains, like HIV-1 in humans, exhibit a marked preference for CD4⁺ CCR5⁺ memory cells, in contrast to the acutely pathogenic SHIV strains which principally target naïve cells (48).Live, attenuated strains of SIV have provided the strongest vaccine protection by far against SIV challenge. Although clinical use of a live, attenuated HIV vaccine is not being considered, understanding the basis of the strong protection afforded by live, attenuated SIV strains remains an important research objective for the insights that can be provided. Most of the attenuated SIV strains that have been used lack a functional nef gene (16, 31, 58, 67). Shacklett et al. (56) used an attenuated SIV strain with modifications in the gp41 transmembrane protein for protection. Here, we describe strong vaccine protection by a replication-competent SIV strain lacking 100 amino acids from the essential gp120 envelope protein in the absence of overtly robust immune responses.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

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.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Derivation and Characterization of a Simian Immunodeficiency Virus SIVmac239 Variant with Tropism for CXCR4

Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4⁺ CXCR4⁺ CCR5⁻ SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4⁻ SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4⁻ SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.Simian immunodeficiency viruses (SIVs) share many structural and biological features with human immunodeficiency virus (HIV), including target cell entry via interactions of the viral envelope glycoprotein (Env) with CD4 and a chemokine coreceptor. For HIV, the most important coreceptors in vivo are CCR5 (2, 13, 19, 21, 22) and CXCR4 (30). HIV type 1 (HIV-1) strains that use only CCR5 (R5 viruses) predominate during the early stages of infection and are critical for transmission (84, 90), as evidenced by the finding that individuals lacking a functional CCR5 protein due to a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (16, 54, 82). Although R5 viruses generally persist in late-stage disease, viruses that can use CXCR4, either exclusively (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of subtype B-infected individuals (15, 43). This coreceptor switch is associated with a more rapid decline in peripheral blood CD4⁺ T cells and a faster progression to AIDS (15, 43, 77), although it is unclear if CXCR4-using viruses are a cause or a consequence of progressing immunodeficiency. Like HIV, the vast majority of SIVs use CCR5 to establish infection (11, 12, 45). However, although CXCR4-using SIVs have been reported (47, 52, 65, 68, 69), their occurrence is rare, especially in models of pathogenic infection, where only one CXCR4-using SIV has been identified (17, 60, 71).This paucity of CXCR4-using SIVs is surprising for several reasons. First, SIV Envs tend to be more promiscuous than HIV-1 Envs and frequently use alternative coreceptors in addition to CCR5, including GPR1, GPR15, CXCR6, and CCR8 (20, 27, 29, 80, 81, 92) but not CXCR4. Second, HIV-2, which is more closely related to SIVmac than to HIV-1 (56, 57), commonly uses CXCR4 in vitro and in vivo (3, 28, 33, 58, 59, 67). Third, rhesus CXCR4 is ∼98% identical to human CXCR4 in amino acid sequence and can function as a coreceptor for HIV-1 in vitro (12). Finally, chimeric simian-human immunodeficiency viruses (SHIVs) that contain X4 HIV Envs on an SIV core can replicate to high levels in vivo and cause disease in rhesus macaques (39, 86). Moreover, it was recently shown that coreceptor switching can occur in rhesus macaques infected with an R5 SHIV (35). Thus, there does not appear to be any block per se against the use of rhesus CXCR4 as an entry coreceptor either in vitro or in vivo, suggesting that SIV is less capable of adapting to use CXCR4 and/or that mutations required for CXCR4 utilization may lead to a virus that is less fit and/or more susceptible to immune control in this host.For HIV-1, the Env determinants for CXCR4 use have been well documented and often involve the acquisition of positively charged amino acids in the V3 loop (18, 32, 87), particularly at positions 11, 24, and 25 (6, 18, 31, 32, 38, 75). Although the SIVmac239 V3 loop is a critical determinant for Env-coreceptor interactions (44, 63, 72), attempts to create an X4 SIVmac239 by introducing positively charged residues into the V3 loop (63) or by inserting a V3 loop from X4 HIV-1 (44) have been unsuccessful. SIVmac155T3, the only CXCR4-using variant of SIVmac that has been identified to date, was isolated from a rhesus macaque with advanced disease and contains additional positively charged residues in V3, although the determinants for CXCR4 use have not been determined (60, 71).Given questions concerning the possible determinants for and/or barriers to coreceptor switching in SIV, we sought to derive a CXCR4-using variant of the well-characterized pathogenic R5 SIV clone SIVmac239. Here we show that SIVmac239 could indeed acquire CXCR4 utilization when it was adapted in vitro for high-efficiency replication in the CXCR4⁺ CCR5⁻ human SupT1 cell line. An env clone from this virus could use CXCR4 in cell-cell fusion and reporter virus infection assays and conferred CXCR4 tropism to a replication-competent SIV. Although V3 mutations were important for CXCR4 use, an L328W change at the V3 crown rather than the acquisition of positively charged residues was required, as was an unusual K47E mutation in the conserved C1 domain of gp120. These changes also caused the highly neutralization-resistant SIVmac239 strain to become more neutralization sensitive to sera and plasmas from SIVmac239-infected animals, and particularly to soluble CD4. These results indicate that mutations distinct from those typically seen for HIV-1 may be required for SIVmac to gain CXCR4 utilization and suggest that these changes render this virus more susceptible to humoral immune control. Collectively, our findings indicate that there are likely to be strong viral and host selection pressures against CXCR4 use that may contribute to the paucity of X4 coreceptor switching for SIVmac in vivo.     

Compartmentalization and Clonal Amplification of HIV-1 Variants in the Cerebrospinal Fluid during Primary Infection

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is a severe neurological disease that affects a subset of HIV-1-infected individuals. Increased compartmentalization has been reported between blood and cerebrospinal fluid (CSF) HIV-1 populations in subjects with HAD, but it is still not known when compartmentalization arises during the course of infection. To assess HIV-1 genetic compartmentalization early during infection, we compared HIV-1 populations in the peripheral blood and CSF in 11 primary infection subjects, with analysis of longitudinal samples over the first 18 months for a subset of subjects. We used heteroduplex tracking assays targeting the variable regions of env and single-genome amplification and sequence analysis of the full-length env gene to identify CSF-compartmentalized variants and to examine viral genotypes within the compartmentalized populations. For most subjects, HIV-1 populations were equilibrated between the blood and CSF compartments. However, compartmentalized HIV-1 populations were detected in the CSF of three primary infection subjects, and longitudinal analysis of one subject revealed that compartmentalization during primary HIV-1 infection was resolved. Clonal amplification of specific HIV-1 variants was identified in the CSF population of one primary infection subject. Our data show that compartmentalization can occur in the central nervous system (CNS) of subjects in primary HIV-1 infection in part through persistence of the putative transmitted parental variant or via viral genetic adaptation to the CNS environment. The presence of distinct HIV-1 populations in the CSF indicates that independent HIV-1 replication can occur in the CNS, even early after HIV-1 transmission.Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to neurological disease in a subset of HIV-infected individuals and may include the development of HIV-1-associated dementia (HAD) (2, 18). HAD is characterized by severe neurological dysfunction, and affected individuals generally have impaired cognitive and motor functions. HIV-1 enters the CNS during primary infection, most likely via the migration of infected monocytes and lymphocytes across the blood-brain barrier (33, 37, 42). The main cell types in the CNS that HIV-1 can productively infect are the perivascular macrophages and microglial cells, which express low receptor densities of CD4, CCR5, and CXCR4 (7, 18, 60, 63). Previous studies have also reported that neurotropic HIV-1 variants are generally macrophage tropic (19, 20, 32, 45, 52, 61). Although cells in the CNS may be infected with HIV-1 during the course of disease, it is still unclear whether productive HIV-1 replication occurs in the CNS early during infection.Genetically compartmentalized HIV-1 variants have been detected in the brains of HAD subjects at autopsy (13, 14, 43, 48, 52) and in the cerebrospinal fluid (CSF) of HAD subjects sampled over the course of infection (26, 46, 51, 59). Extensive compartmentalization between the periphery and the CNS has been reported in subjects with HAD; however, it is not yet known when compartmentalization occurs during the course of HIV-1 infection. Primary HIV-1 infection refers to the acute and early phases of infection, during which peak plasma viremia often occurs and a viral “set point” may be reached (8, 34), within the first year after HIV exposure (64). Studies examining compartmentalization between the blood plasma and CSF during primary infection have been limited, and extensive compartmentalization has not been detected in primary infection subjects (26, 50).In this study, we examined HIV-1 genetic compartmentalization between the peripheral blood and CSF during primary HIV-1 infection. Cross-sectional and longitudinal blood plasma and CSF samples were analyzed for viral compartmentalization using the heteroduplex tracking assay (HTA) and single genome amplification (SGA). We used the HTA to differentiate between HIV-1 variants in the CSF that were either compartmentalized to the CSF or equilibrated with the peripheral blood. Previous studies have used the HTA to separate HIV-1 genetic variants in different anatomical compartments (10, 24, 27, 51) and to follow HIV-1 evolutionary variants over the course of infection (9, 25, 31, 41, 49, 50). We also conducted SGA on a subset of subjects to further examine viral genetic compartmentalization during primary infection. Here we report the detection of compartmentalized and clonally amplified HIV-1 variants in the CSF of subjects in the primary stage of HIV-1 infection. Our results suggest that minor to extensive HIV-1 genetic compartmentalization can occur between the periphery and the CNS during primary HIV-1 infection and that viral compartmentalization, as measured in the CSF, is transient in some subjects.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.