The requirement by HIV-1 for specific cellular interacting factors during the entry step offers an opportunity for the development of vaccines and drugs that target this crucial step in the virus replication cycle (Dhami et al., 2009
; Hunt and Romanelli, 2009
; Pantophlet and Burton, 2006
; Phogat et al., 2007
). Coreceptors play an important role in initiating infection at the cellular level. Additionally, coreceptor utilization is an important determinant of the rate of disease progression. The emerging availability of entry inhibitors such as the CCR5 antagonists underlines the importance of better characterization of coreceptor utilization and cellular tropism by HIV-1 isolates particularly in heavily burdened countries where the drugs are likely to be required on a large scale for the clinical management of HIV/AIDS. In this study, we generated 35 full-length env
clones from seven dualtropic isolates of HIV-1 subtype C, in order to determine whether they were a mixture of CCR5 and CXCR4 quasispecies or dualtropic viruses at the clonal level. We also interrogated the sequence characteristics of these clones in order to better elucidate the genetic determinants of coreceptor utilization by HIV-1 subtype C viruses. We found that CXCR4-tropic clones dominated within the dualtropic viral isolates quasispecies. A minority proportion of dualtropic clones were also identified. Unexpectedly, we found that there was not a single CCR5-monotropic env
clone from the seven primary isolates analyzed in this study. This is an unusual finding considering that many studies have shown that HIV-1 subtype C viruses even in late stages of disease utilize CCR5-only predominantly for cell entry. We thus expected to find a significant proportion of the remnants of these viruses among the quasispecies of the dualtropic isolates. Instead, all the clones detected in this study used CXCR4 as the coreceptor for cell entry, with a minority of these (14.3%) also able to mediate entry via the CCR5 receptor. Our results may explain why in previous studies of some of the dualtropic isolates described here (CM9, SW20 and SW30); the isolates could be strongly inhibited by CXCR4 inhibitors but only modestly by CCR5 inhibitors (Cilliers et al., 2003
). These earlier results can now be explained by the observation that although these isolates are dualtropic, they are dominated by X4 variant clones.
An alternative explanation of our findings is that these isolates changed their coreceptor preference during in vitro passages in PBMC co-cultures as has been previously described (Voronin et al., 2007
). This possible explanation is supported by the finding that isolates CM9 and SW30 displayed remarkably lower CCR5 utilization capacity () than was previously described (Cilliers et al., 2003
). It is also worth noting that although isolates BW17, SW20 and Du36_5 showed a possible bias towards CCR5 utilization and were clearly dualtropic, all the env
molecular clones generated from these isolates by bulk PCR amplification were CXCR4-only using. This finding strongly suggested that the bulk PCR could be biased towards X4 viruses. We therefore performed limiting endpoint dilution PCR on one dualtropic viral isolate (Du36_5) which was biased towards CCR5 utilization (). Remarkably, of 26 functional env
clones generated by this approach, 24 exhibited dualtropism, one used CXCR4 exclusively and one used CCR5 exclusively. We therefore conclude that dualtropic HIV-1 subtype C isolates are dominated by X4 and X4R5 clones with negligible proportion of R5 monotropic clones.
It has been recently proposed that coreceptor switching is associated with deleterious mutations in env
that diminish CCR5-tropism as mutations associated with CXCR4 utilization accumulate (Coetzer et al., 2008
). Although we did not directly test for coreceptor binding in this study, our results are consistent with the proposal by Coetzer et al. and with their observation that coreceptor switching is associated with a rapid decrease in the ability to use CCR5. Our results may suggest that in HIV-1 subtype C, the mutations required for adaptation to CXCR4 utilization significantly reduce the ability of env
to utilize CCR5, thus resulting in reduced fitness of CCR5 utilizing viruses. This could in turn lead to the selection and amplification of clones able to utilize CXCR4. We can speculate that given the low frequency of HIV-1 subtype C CXCR4 utilizing viruses reported in various studies, more accumulated mutations are required for switching to CXCR4 utilization for this subtype. Alternatively, the changes required for a switch to CXCR4 utilization may result in a bigger fitness deficit for HIV-1 subtype C CCR5-tropic variants thus leading to selection against these viruses once adaptation to CXCR4 utilization has been accomplished. Further studies will be required to carefully investigate the specific localization and nature of complementary mutations required for HIV-1 subtype C env
We also investigated the genetic characteristics associated with CXCR4-usage or dualtropism for HIV-1 subtype C viruses. Our results may be limited by founder effects since we could not generate R5 sequences from the study isolates but we nevertheless had HIV-1 subtype C R5 sequences available from the Los Alamos database that facilitated this comparative analysis. As described for HIV-1 subtype B, the subtype C third variable loop of gp120 (V3 region) is a major determinant of whether CXCR4 or CCR5 will be the accessory protein used by the virus for membrane fusion (Cilliers et al., 2003
; Coetzer et al., 2007
; Coetzer et al., 2006
; Fouchier et al., 1992
; Morris et al., 2007
; Ndung’u et al., 2006
). Typically, the V3 region consists of approximately 35 amino acids in CCR5-tropic viruses (Coetzer et al., 2006
). Consistent with earlier studies, we found that V3 loop amino acid characteristics are important determinants of coreceptor tropism. In most cases, we found that the V3 loop crown of CXCR4-utilizing clones had basic amino acid substitutions which differed from the canonical GPGQ sequence found in CCR5 HIV-1 subtype C viruses to GPGX (where X is any other amino acid), GRGH, GPGR or GPRY. X4 variants are more variable than R5 viruses in the V3 region particularly at positions 11 and 25 which tend to be mostly positively charged amino acids, often arginine (R), lysine (K) or histidine (H). We found the presence of a basic amino acid at both or one of these positions for the majority of clones in this study (85%). In addition, in X4 variants there may be insertions particularly between positions 13 and 14 of the V3 loop contributing to an increased length. We found amino acid insertions in 71% of clones in this study. X4 variants may also be distinguished from R5 viruses as they usually have an increased net V3 charge. Consistent with these observations, we found that 100% of X4-utilizing clones had V3 loop amino acid charges of +5 or more.
The V3 region however, is not the exclusive determinant of coreceptor usage and other regions within the env
gene may also contribute to viral tropism. The V1/V2 and V4/V5 regions have been implicated in playing a role in determining the biological phenotype of the virus. Specifically, the number of N-linked carbohydrate moieties in these variable loops has been associated with coreceptor determination (Chohan et al., 2005
; Coetzer et al., 2008
; Masciotra et al., 2002
; Pollakis et al., 2001
). Here we found a strong association between the number of N-linked glycosylation sites and coreceptor utilization with X4 clones having a significantly higher number of these sites than R5 clones from the database overall and in the V1/V2 or V4/V5 regions (). In contrast in the V3 region, the number of sites was significantly higher in R5 sequences than X4/X4R5 sequences (). A previous longitudinal study of HIV-1 env
evolution showed no significant changes in N-linked glycosylation sites of 23 viral isolates from 5 patients followed for 2-4 years (Coetzer et al., 2007
). Therefore our findings may suggest a rapid accumulation of N-linked glycosylation sites as coreceptor tropism switches, as opposed to a slow accumulation of these sites over time. This is consistent with recent findings of rapid decline in CCR5 utilization as alternate coreceptor utilization emerges in HIV-1 subtype B infection (Coetzer et al., 2008
). In both HIV-1 subtypes A and C, shorter V1/V2 loop sequences and fewer predicted N-linked glycosylation sites have been correlated with preferential heterosexual viral transmission (Chohan et al., 2005
; Derdeyn et al., 2004
). We did not find significant differences in V1/V2 length between R5 and X4 clones in this study but a trend towards shorter V4/V5 for X4 clones was noted (). Further longitudinal studies will be necessary in order to better understand HIV-1 subtype C transmission, coreceptor switching and the env
genetic characteristics associated with these processes. Overall, our results suggest that sequence characteristics in the V3 loop, the V4/V5 loop length as well as the number of env
predicted N-linked glycosylation sites are the primary genotypic determinants for viral tropism in HIV-1 subtype C.
It is worth noting that we did not perform limiting endpoint dilution of samples in this study except for isolate Du36_5. Therefore we cannot completely rule out the presence of substantial frequencies of R5-monotropic viruses in the quasispecies of the isolates where endpoint dilution was not used. However, the absence of these clones in bulk amplified clones, in CCR5 only expressing cells and in endpoint diluted Du36_5 isolate that is biased towards CCR5 is all suggestive of absence of such quasispecies or presence at very low frequency. Our results appear to contradict the recent findings of Irlbeck et al. (2008)
but it must be emphasized that in that study, samples were analyzed directly from plasma in contrast to our study in which we examined in vitro propagated isolates. Further studies will be needed to determine whether env
clones directly obtained from patients with dualtropic HIV-1 subtype C viruses have a bias towards CCR5 or CXCR4 tropism.
In conclusion, we show in this study that dualtropic viral isolates consist of predominantly X4 and X4R5 clones. Thirty of 35 env clones analyzed from PBMCs utilized X4 only as the coreceptor for entry into cells, whereas 5 of 35 clones tested displayed dualtropism and no CCR5-only utilizing clones were identified. R5 monotropic clones could not be detected even when the isolates were cultured in cells expressing CCR5 coreceptor only. We also failed to detect a significant number of R5 monotropic clones when we changed our approach of viral amplification from bulk PCR to limiting endpoint dilution PCR for one dualtropic isolate showing bias towards CCR5 tropism. Viral env sequences from both CXCR4 and CCR5-expressing cells were indistinguishable and possessed X4/dualtropic characteristics. Furthermore, we describe env sequence characteristics associated with CXCR4 utilization in HIV-1 subtype C. In addition to sequence changes in the env V3 region, we identify the number of N-linked glycosylation sites in the V1/V2, V3 and V4/V5 regions as major determinants of coreceptor utilization in HIV-1 subtype C. We also show that the length of the V4/V5 is a possible determinant of coreceptor utilization. We note that our results are consistent with recent findings of the rapid loss of fitness of CCR5 envelope as coreceptor switching emerges and suggest that the sequence characteristics associated with coreceptor switch must occur rapidly in vivo. Further studies are needed to better characterize coreceptor switching, particularly in the context of HIV-1 subtype C, the predominant subtype in the world. We have generated 35 full-length CXCR4- or dualtropic clones of HIV-1 subtype C, important reagents that will facilitate further functional studies of this globally predominant subtype. Our results have important implications for coreceptor antagonist design and application, and further contribute to better understanding of HIV-1 pathogenesis.