In this study of sequences from eight individuals with HIV-1 subtype A infection, we showed that modifications within the V1-V5 envelope segments confer increased replication capacity over the course of infection (Fig. ). Previous studies with subtype B HIV-1 have also demonstrated that ex vivo replication capacity increases over the course of infection (4
). Similarly, in the simian immunodeficiency virus/macaque model, longitudinally collected viruses demonstrate greater in vivo replication than variants isolated from early in infection (31
). We also showed that longitudinally isolated V1-V5 envelope segments conferred significantly increased resistance to CCR5 inhibitors, TAK779 and PSC-RANTES (Fig. ). In addition, we demonstrated that in the majority of subjects, V1-V5 domains from the chronic phase of infection, compared to those from early after HIV-1 acquisition, led to increased replication capacity in cells with low CCR5 densities (Fig. ). From these significantly correlated measures of CCR5 use (Fig. ), we concluded that viruses from the chronic phase of infection are more efficient at utilizing CCR5 than variants isolated early after infection. Among the longitudinally collected variants from the different subjects, we also found that resistance to fusion inhibitors increases over time, suggesting that chronic-infection viruses are more fusogenic than isolates from early in infection (Fig. ). Previous studies and our controls suggest that these measurements could be surrogate markers for coreceptor affinity and fusion kinetics (59
). Because cell entry is potentially the rate-limiting step in HIV-1 replication (3
), in the aggregate, our data suggest that increases in replication capacity over the course of infection are potentially related to greater affinity for the CCR5 receptor and/or faster fusion kinetics.
A previous study has suggested that acute- and chronic-infection variants are not significantly different in their sensitivities to CCR5 and fusion inhibitors (73
). In that study, however, the virus isolates from the acute and chronic phases of infection were obtained from different subjects. In contrast to that publication and similar to our results, other studies which have examined longitudinally collected variants have suggested that viruses become more resistant to CCR5 and fusion inhibitors over time (27
). In these studies, PBMC cocultures were used to demonstrate that viruses from the chronic phase of infection have differential susceptibility to receptor and fusion inhibitors compared to late-stage variants. In contrast to these studies, we examined subtype A viruses as opposed to the presumably subtype B HIVs examined in the previous publications. In addition, we avoided in vitro adaptation that may occur among viruses in PBMC cocultures by amplifying V1-V5 envelope segments and incorporating them into a full-length HIV-1 clone using yeast gap repair methodology. One of the major differences between our study and previous publications, however, is that we examined differences among variants from the early and chronic phases of infection as opposed to comparing viruses from the chronic and late stages of disease. Collectively, these studies suggest that both subtype A and B HIVs found early in infection are extremely sensitive to CCR5 antagonists and fusion inhibitors, and sensitivity to these compounds progressively decreases over later times in infection. Studies from our group and others demonstrate that there is a strong correlation between sensitivity to TAK779 and clinically relevant CCR5 antagonists, such as maraviroc and vicriviroc (81
; T. Henrich, M. Sagar, and D. Kuritzkes, unpublished data). Potential implications from these studies are that chemokine antagonists and fusion inhibitors may be ideal therapeutic drugs early in infection. In addition, although CCR5 utilization increases over time, HIV-1 infection starts with variants highly sensitive to CCR5 inhibitors, which further justifies the exploration of CCR5 inhibition as a potential means to interrupt transmission (36
Another major difference between our work and the majority of previously published studies is that we examined envelope phenotypic differences using replication competent viruses as opposed to viral pseudotypes. We employed a modified yeast gap repair homologous recombination system to generate a large number of recombinant replication-competent viruses (Fig. ). Within the HIV field, the majority of studies examining envelope glycoprotein phenotypic differences employ virus pseudotypes. Although, pseudoviruses are highly conducive to high-throughput analysis of a large number of envelopes, there are a number of inherent limitations. First, the virus pseudotypes are restricted to a single round of replication, and thus, phenotypic differences conferred by the viral envelope glycoproteins, such as replication capacity, cannot be examined over multiple replication cycles. Second, pseudoviruses often display different phenotypes compared to replication-competent viruses. For instance, single-cycle pseudotypes compared to replication-competent chimeric viruses have demonstrated different sensitivities to CCR5 inhibitors on the same target cells (64
). Third, the number of envelope glycoproteins expressed on a viral particle may be different among pseudoviruses versus replication-competent viruses (60
). This may relate to differences in the number of defective envelopes and glycoprotein processing among the two types of recombinant viruses. Envelope glycoprotein properties need to be examined in more detail among replication-competent recombinant viruses and viral pseudotypes to definitely document the differences among these two virus constructs.
The exact biological mechanism for increased CCR5 utilization among chronic-stage viruses compared to variants early after infection remains unclear. Although, TAK779 is an allosteric (2
) and not a competitive inhibitor, TAK779 sensitivity correlates with affinity for the CCR5 receptor (66
). Furthermore, TAK779 IC50
s correlate with sensitivity to the CCR5 competitive inhibitor RANTES (Fig. ) (40
). Thus, we suggest that increased CCR5 usage is because of a greater affinity for the CCR5 receptor. Another potential mechanism for higher CCR5 utilization is that chronic-stage envelopes could bind a broader array of CCR5 conformations. Indeed, natural CCR5 ligands, such as RANTES, can trigger internalization, as well occupy and presumably distort the receptor (54
). Thus, in vivo, the CCR5 receptor may have different conformations in the presence of chemokines. It has been shown that envelopes from the late phase of infection compared to the variants from the chronic stage of disease display an increased ability to bind a broad range of chimeric CCR5 receptors (30
). Therefore, an ability to bind different structural forms of the CCR5 receptor may explain the increased CCR5 usage among chronic-stage variants compared to the early-infection isolates. Finally, it also possible that chronic- and early-stage viruses may have similar binding to the CCR5 receptor, but after CCR5 attachment, viruses with chronic- versus early-stage V1-V5s may have a higher propensity for proceeding to fusion; this may account for the differences in CCR5 utilization. Previous studies have suggested that CCR5 affinity is directly correlated with fusion capacity (66
), and thus viruses with chronic- versus early-infection V1-V5s may possess both greater affinity for the CCR5 receptor and increased fusion capacity. Thus, our studies cannot distinguish whether increased replication capacity among the chronic-stage variants is due to increased CCR5 utilization and/or faster fusion kinetics.
Interestingly, both sensitivity to CCR5 inhibitors and differential fusion capacity have been previously mapped to sequence changes within the envelope V3 loop and the bridging sheet, which are important for coreceptor binding (16
). Among our eight subjects, differences among the early- and chronic-infection envelope sequences were evident primarily in the V3 loop and not in the bridging sheet, which consists of β strands 2, 3, 20, and 21 (35
). V3 loop modifications, however, are not solely responsible for the phenotypic differences observed among early- and chronic-infection envelopes. Our chimeric envelope data suggest that sequence changes within the V3 loop in conjunction with differences in the V1-V2 loops influence CCR5 usage (Fig. ). Furthermore, although each envelope harbored an isogenic transmembrane domain, notable differences were observed in the sensitivity to fusion inhibitor T-20 among early- and chronic-infection V1-V5 segments, and our envelope chimera data suggest that changes within the V1-V3 domains also affected this phenotype (Fig. ). Because of numerous and diverse changes in the V1-V3 domains between the early- and chronic-infection envelope isolates, we were unable to identify canonical envelope modifications as being responsible for the enhanced CCR5 utilization and fusion capacity. Interestingly, previously identified polymorphisms, such as changes at positions 318 and 319 of the V3 loop (HXB2 numbering), which have been associated with differential sensitivity to CCR5 inhibitors (40
), were highly conserved among early- and chronic-infection envelope sequences. The 318/319 consensus tyrosine (Y)/alanine (A) motif was modified to serine (S)/A in QA284-8C, Y/threonine (T) in QA779-5C and QA77913C, and Y/glycine (G) among all QC449 variants. Thus, in our study, these exclusive 318/319 modifications within the V3 loop did not influence the majority of observed changes in CCR5 usage. Our studies contrast with other publications potentially because of the differences in the subtype of the virus and because we examined longitudinally isolated viral variants from natural infection as opposed to laboratory-derived viral strains.
Progressively decreasing sensitivity to CCR5 entry inhibitors provides insight into the selection mechanisms acting on the virus during the course of infection within a host. The host antibody neutralizing response is a well-described selection force that drives evolution in the HIV-1 envelope gene (10
). Our results imply that receptor use may be another potential selection mechanism driving changes in the viral envelope glycoprotein. After HIV-1 acquisition, a large percentage of memory CD4+
T cells are eliminated from the mucosal tissues (5
). In addition, CCR5 receptor levels on the remaining target cells may be downregulated through high-level expression of chemokines such as RANTES, MIP-1α, and MIP-1β (1
). Both processes likely decrease the availability of CD4+
T cells with high levels of the CCR5 receptor. The dearth of these cell types potentially forces HIV-1 to evolve envelopes with an increased ability to use low levels of the CCR5 receptor and/or switch to CXCR4 use late in infection. The need to evolve a greater ability to use low levels of CCR5 is likely especially true for subtype A HIV-1, where use of other coreceptors, such as CXCR4, has been infrequently documented (26
). It should be noted, however, that not all individuals, such as QB424, displayed an enhanced ability to use CCR5 over time, suggesting that the potential selection forces or virus responses may be different in some hosts.
Chronic-stage viruses showed increased CCR5 utilization compared to early-phase viruses even though envelope glycoproteins expand variable loops and increase the number of glycosylated amino acids. Similar to our previous investigations with the same subjects (76
), we found that chronic-stage envelopes had a significantly higher number of glycosylated residues than viruses early after HIV-1 acquisition. Envelope variable loop expansion and increased glycosylation likely evolve to conceal conserved antigenic portions on the viral envelope glycoprotein, such as the CD4 and/or the coreceptor binding site (35
), from the host neutralizing antibody response. Shielding these domains, however, may hinder access of the viral envelope glycoprotein for the host cell receptors. Our results imply that increased glycosylation did not adversely affect CCR5 binding. Interestingly, we also found no difference either in sensitivity to a CD4 inhibitor, soluble CD4, or in replication capacity in cells with limiting levels of the CD4 receptor among chronic- and early-stage envelope V1-V5 segments (Fig. and ). It has been hypothesized that HIV-1 envelopes with an increased ability to use low levels of CD4 evolve in the absence of humoral immune pressure, such as in the central nervous system, an immunologically privileged site (18
). Our data suggest that the converse does not necessarily hold, because neutralizing antibodies, which we have previously documented in these subjects (76
), do not lead to envelope modifications that decrease the efficiency of CD4 receptor usage.
After observing increased CCR5 utilization among chronic-stage envelopes, we hypothesized that envelopes from late in infection were more likely to replicate in primary cells with limiting levels of CCR5, such as macrophages. Indeed, some previous studies suggest that isolates from late in disease are more macrophage-tropic than those from early in infection (23
). Furthermore, it has been suggested that higher CCR5 binding confers macrophage tropism (22
). We, however, observed no significant differences among early envelopes versus chronic-stage variants in their ability to replicate in MDMs (Fig. ). Therefore, our observations support previous conclusions that replication capacity in macrophages does not correlate with an ability to use CCR5 (59
). Because we did not observe significant differences in CD4 receptor use, however, we cannot directly corroborate that CD4 affinity predominately influences macrophage tropism as has been previously suggested (59
In the same subjects analyzed in this study, we have previously shown that changes within the V1-V2 envelope loops confer significantly increased neutralization resistance to autologous plasma (76
). Although we did not directly assess neutralization sensitivity of the chimeric viruses in this study to autologous plasma, collectively our results imply that evolution within the envelope glycoprotein over the course of infection that occurs in response to the host antibody response does not necessarily confer a fitness cost in terms of receptor usage and replication capacity. It should be noted, however, that effects on entry and replication of the specific modifications that confer neutralization escape will need to be examined in detail to validate this hypothesis. In summary, our data suggest that modifications within the envelope V1-V3 lead to antibody neutralization escape, an increased ability to utilize the CCR5 receptor, and faster fusion kinetics.