R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIVSF33A Molecular Clones

In vivo adaptation of simian-human immunodeficiency virus (SHIV) clone SHIV(SF33) resulted in the emergence of pathogenic isolate SHIV(SF33A), which caused a rapid and severe CD4(+) T-cell depletion when inoculated into rhesus macaques. Two molecular clones generated by inserting the env V1-to-V5 region amplified from SHIV(SF33A)-infected animals into the parental SHIV(SF33) genome retained a pathogenic phenotype. The gp120 envelope glycoproteins of pathogenic clones SHIV(SF33A2) and SHIV(SF33A5) conferred a threefold increase in viral entry and fusogenicity compared to the parental glycoprotein. Changes in gp120 were also responsible for a higher replication capacity and cytopathicity in primary CD4(+) T-cell cultures. Last, gp120 carried the determinants of SHIV(SF33A) neutralization resistance. Thus, changes in SHIV(SF33A) gp120 produced a set of properties that could account for the pathogenic phenotype observed in vivo. Measurement of antibody binding to SHIV(SF33A) viral particles revealed an increased exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody in a region that was shown to contribute to coreceptor binding. Exposure of this epitope occurred in the absence of CD4 binding, suggesting that the envelope glycoprotein of pathogenic SHIV(SF33A) clones folded in a conformation that was primed for interaction with CXCR4 or for the subsequent step of fusion.     

Adoption of an "open" envelope conformation facilitating CD4 binding and structural remodeling precedes coreceptor switch in R5 SHIV-infected macaques

A change in coreceptor preference from CCR5 to CXCR4 towards the end stage disease in some HIV-1 infected individuals has been well documented, but the reasons and mechanisms for this tropism switch remain elusive. It has been suggested that envelope structural constraints in accommodating amino acid changes required for CXCR4 usage is an obstacle to tropism switch, limiting the rate and pathways available for HIV-1 coreceptor switching. The present study was initiated in two R5 SHIV(SF162P3N)-infected rapid progressor macaques with coreceptor switch to test the hypothesis that an early step in the evolution of tropism switch is the adoption of a less constrained and more "open" envelope conformation for better CD4 usage, allowing greater structural flexibility to accommodate further mutational changes that confer CXCR4 utilization. We show that, prior to the time of coreceptor switch, R5 viruses in both macaques evolved to become increasingly sCD4-sensitive, suggestive of enhanced exposure of the CD4 binding site and an "open" envelope conformation, and this correlated with better gp120 binding to CD4 and with more efficient infection of CD4(low) cells such as primary macrophages. Moreover, significant changes in neutralization sensitivity to agents and antibodies directed against functional domains of gp120 and gp41 were seen for R5 viruses close to the time of X4 emergence, consistent with global changes in envelope configuration and structural plasticity. These observations in a simian model of R5-to-X4 evolution provide a mechanistic basis for the HIV-1 coreceptor switch.     

Different mutational pathways to CXCR4 coreceptor switch of CCR5-using simian-human immunodeficiency virus

We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N))-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIV(CA28NP)) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIV(CA28NL)) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIV(SF162P3N) isolate in vivo.     

Induction of CD8+ cells able to suppress CCR5-tropic simian immunodeficiency virus SIVmac239 replication by controlled infection of CXCR4-tropic simian-human immunodeficiency virus in vaccinated rhesus macaques

Recent recombinant viral vector-based AIDS vaccine trials inducing cellular immune responses have shown control of CXCR4-tropic simian-human immunodeficiency virus (SHIV) replication but difficulty in containment of pathogenic CCR5-tropic simian immunodeficiency virus (SIV) in rhesus macaques. In contrast, controlled infection of live attenuated SIV/SHIV can confer the ability to contain SIV superchallenge in macaques. The specific immune responses responsible for this control may be induced by live virus infection but not consistently by viral vector vaccination, although those responses have not been determined. Here, we have examined in vitro anti-SIV efficacy of CD8+ cells in rhesus macaques that showed prophylactic viral vector vaccine-based control of CXCR4-tropic SHIV89.6PD replication. Analysis of the effect of CD8+ cells obtained at several time points from these macaques on CCR5-tropic SIVmac239 replication in vitro revealed that CD8+ cells in the chronic phase after SHIV challenge suppressed SIV replication more efficiently than those before challenge. SIVmac239 superchallenge of two of these macaques at 3 or 4 years post-SHIV challenge was contained, and the following anti-CD8 antibody administration resulted in transient CD8+ T-cell depletion and appearance of plasma SIVmac239 viremia in both of them. Our results indicate that CD8+ cells acquired the ability to efficiently suppress SIV replication by controlled SHIV infection, suggesting the contribution of CD8+ cell responses induced by controlled live virus infection to containment of HIV/SIV superinfection.     

Coreceptor switch in R5-tropic simian/human immunodeficiency virus-infected macaques

The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in approximately 50% of human immunodeficiency virus type 1 (HIV-1) subtype B-infected individuals as disease advances is not well understood. Among the reasons proposed are target cell limitation and better immune recognition of the CXCR4 (X4)-tropic compared to the CCR5 (R5)-tropic virus. We document here X4 virus emergence in a rhesus macaque (RM) infected with R5-tropic simian/human immunodeficiency virus, demonstrating that coreceptor switch can happen in a nonhuman primate model of HIV/AIDS. The switch to CXCR4 usage in RM requires envelope sequence changes in the V3 loop that are similar to those found in humans, suggesting that the R5-to-X4 evolution pathways in the two hosts overlap. Interestingly, compared to the inoculating R5 virus, the emerging CXCR4-using virus is highly neutralization sensitive. This finding, coupled with the observation of X4 evolution and appearance in an animal with undetectable circulating virus-specific antibody and low cellular immune responses, lends further support to an inhibitory role of antiviral immunity in HIV-1 coreceptor switch.     

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.     

Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.     

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Frequent intrapatient recombination between human immunodeficiency virus type 1 R5 and X4 envelopes: implications for coreceptor switch

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4+ cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Hybrid Approach for Predicting Coreceptor Used by HIV-1 from Its V3 Loop Amino Acid Sequence

Background

HIV-1 infects the host cell by interacting with the primary receptor CD4 and a coreceptor CCR5 or CXCR4. Maraviroc, a CCR5 antagonist binds to CCR5 receptor. Thus, it is important to identify the coreceptor used by the HIV strains dominating in the patient. In past, a number of experimental assays and in-silico techniques have been developed for predicting the coreceptor tropism. The prediction accuracy of these methods is excellent when predicting CCR5(R5) tropic sequences but is relatively poor for CXCR4(X4) tropic sequences. Therefore, any new method for accurate determination of coreceptor usage would be of paramount importance to the successful management of HIV-infected individuals.

Results

The dataset used in this study comprised 1799 R5-tropic and 598 X4-tropic third variable (V3) sequences of HIV-1. We compared the amino acid composition of both types of V3 sequences and observed that certain types of residues, e.g., Asparagine and Isoleucine, were preferred in R5-tropic sequences whereas residues like Lysine, Arginine, and Tryptophan were preferred in X4-tropic sequences. Initially, Support Vector Machine-based models were developed using amino acid composition, dipeptide composition, and split amino acid composition, which achieved accuracy up to 90%. We used BLAST to discriminate R5- and X4-tropic sequences and correctly predicted 93.16% of R5- and 75.75% of X4-tropic sequences. In order to improve the prediction accuracy, a Hybrid model was developed that achieved 91.66% sensitivity, 81.77% specificity, 89.19% accuracy and 0.72 Matthews Correlation Coefficient. The performance of our models was also evaluated on an independent dataset (256 R5- and 81 X4-tropic sequences) and achieved maximum accuracy of 84.87% with Matthews Correlation Coefficient 0.63.

Conclusion

This study describes a highly efficient method for predicting HIV-1 coreceptor usage from V3 sequences. In order to provide a service to the scientific community, a webserver HIVcoPred was developed (http://www.imtech.res.in/raghava/hivcopred/) for predicting the coreceptor usage.     

Effects of partial deletions within the human immunodeficiency virus type 1 V3 loop on coreceptor tropism and sensitivity to entry inhibitors

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1_R3A, which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Δ9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1_89.6. These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.     

Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.     

V3 Loop Sequence Space Analysis Suggests Different Evolutionary Patterns of CCR5- and CXCR4-Tropic HIV

The V3 loop of human immunodeficiency virus type 1 (HIV-1) is critical for coreceptor binding and is the main determinant of which of the cellular coreceptors, CCR5 or CXCR4, the virus uses for cell entry. The aim of this study is to provide a large-scale data driven analysis of HIV-1 coreceptor usage with respect to the V3 loop evolution and to characterize CCR5- and CXCR4-tropic viral phenotypes previously studied in small- and medium-scale settings. We use different sequence similarity measures, phylogenetic and clustering methods in order to analyze the distribution in sequence space of roughly 1000 V3 loop sequences and their tropism phenotypes. This analysis affords a means of characterizing those sequences that are misclassified by several sequence-based coreceptor prediction methods, as well as predicting the coreceptor using the location of the sequence in sequence space and of relating this location to the CD4⁺ T-cell count of the patient. We support previous findings that the usage of CCR5 is correlated with relatively high sequence conservation whereas CXCR4-tropic viruses spread over larger regions in sequence space. The incorrectly predicted sequences are mostly located in regions in which their phenotype represents the minority or in close vicinity of regions dominated by the opposite phenotype. Nevertheless, the location of the sequence in sequence space can be used to improve the accuracy of the prediction of the coreceptor usage. Sequences from patients with high CD4⁺ T-cell counts are relatively highly conserved as compared to those of immunosuppressed patients. Our study thus supports hypotheses of an association of immune system depletion with an increase in V3 loop sequence variability and with the escape of the viral sequence to distant parts of the sequence space.     

Effects of immunization with CCR5-based cycloimmunogen on simian/HIVSF162P3 challenge

A synthetic cycloimmunogen targeting the HIV-1 coreceptor CCR5 was evaluated for its capacity to induce CCR5-specific Abs with anti-HIV-1 activity in cynomolgus macaques. The cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of human CCR5 was chemically prepared, in which the Gly-Glu dipeptide links the amino and carboxy termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (Arg168 to Cys178) of extracellular loop-2 in CCR5. The immunization of cynomolgus macaques with the cDDR5-conjugated multiple-Ag peptide (cDDR5-MAP) induced anti-cDDR5 serum production for approximately 15 wk after the third immunization. The antisera raised against cDDR5-MAP reacted with both human and macaque CCR5s, and potently suppressed infection by the R5 HIV-1 laboratory isolate (HIV JRFL), R5 HIV-1 primary isolates (clade A:HIV 93RW004 and clade C:HIV MJ4), and a pathogenic simian/HIV (SHIV SF162P3) bulk isolate in vitro. To examine the prophylactic efficacy of anti-CCR5 serum Ab for acute HIV-1 infection, cynomolgus macaques were challenged with SHIV SF162P3. The cDDR5-MAP immunization attenuated the acute phase of SHIV SF162P3 replication. The geometric mean plasma viral load in the vaccinated macaques was 217.10 times lower than that of the control macaques at 1 wk postchallenge. Taken together, these results suggest that cDDR5-MAP immunization is an effective prophylactic vaccine strategy that suppresses and delays viral propagation during the initial HIV-1 transmission for the containment of HIV-1 replication subsequent to infection.     

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.     

Pathogenic Consequences of Vaginal Infection with CCR5-Tropic Simian-Human Immunodeficiency Virus SHIVSF162P3N

We previously reported efficient transmission of the pathogenic R5 simian-human immunodeficiency virus SHIV(SF162P3N) isolate in Indian rhesus macaques by intravenous and intrarectal inoculations, with a switch to CXCR4 coreceptor usage in ～50% of infected animals that progressed rapidly to disease. Since women continue to be disproportionately affected by HIV, we developed an animal model based on the intravaginal challenge of female rhesus monkeys with SHIV(SF162P3N) and sought to validate the utility of this model to study relevant aspects of HIV transmission and pathogenesis. The effect of viral dose on infection outcome was evaluated to determine the optimal conditions for the evaluation of HIV-1 preventive and therapeutic strategies. We found that the virus can successfully cross the vaginal mucosal surface to establish infection and induce disease with coreceptor switch, but with lower efficiencies compared to intravenous and rectal transmissions. In contrast to intrarectal infection, peak and cumulative viral load over a 1 year-infection period were significantly greater in macaques exposed intravaginally to lower rather than higher inoculum doses. Moreover, low and transient viremia was observed only in macaques that were challenged intravaginally twice within the same day with a high dose of virus, which can be seen as doubling the dose. Taken together, these results show that SHIV(SF162P3N) can successfully transmit across the genital mucosa, undergo coreceptor switch, and induce disease. However, the administered dose appears to impact SHIV(SF162P3N) vaginal infection outcome in an unexpected manner.     

Fitness disadvantage of transitional intermediates contributes to dynamic change in the infecting-virus population during coreceptor switch in R5 simian/human immunodeficiency virus-infected macaques

Fitness disadvantage of the transitional intermediates compared to the initial R5 viruses has been suggested to constitute one of the blockades to coreceptor switching, explaining the late appearance of X4 viruses. Using a simian model for human immunodeficiency virus type 1 (HIV-1) coreceptor switching, we demonstrate in this study that similar molecular evolutionary pathways to coreceptor switch occur in more than one R5 simian/human immunodeficiency virus (SHIV)(SF162P3N)-infected macaque. In infected animals where multiple pathways for expansion or switch to CXCR4 coexist, fitness of the transitional intermediates in coreceptor usage efficiency influences their outgrowth and representation in the infecting virus population. Dualtropic and X4 viruses appear at different disease stages, but they have lower entry efficiency than the coexisting R5 strains, which may explain why they do not outcompete the R5 viruses. Similar observations were made in two infected macaques with coreceptor switch, providing in vivo evidence that fitness disadvantage is an obstacle to X4 emergence and expansion.     

Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences

Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3(+) T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequence-based "virtual phenotyping" are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.