In this paper we have described the construction and biological analysis of the first infectious proviral DNA molecular clone of HIV-1 subtype C from the African continent. This clone is a chimeric molecule, because the majority of env
was derived from sample 96MOLE1 while the rest of the genome was from sample 96BW06. This clone is derived from primary viral isolates, because the viruses were not propagated in immortalized cell lines prior to DNA amplification. MJ4 utilizes CCR5 as the coreceptor for entry into susceptible cells, consistent with observations from other studies that field isolates of HIV-1 subtype C viruses mostly use this coreceptor for cell entry. In addition, most viruses that are transmitted from individual to individual are known to be macrophage-tropic and thus CCR5 using (10
). This clone may be important in elucidating genetic determinants that underlie HIV-1 subtype C disease pathogenesis, such as its apparent failure to evolve to use other chemokine receptors to mediate cell entry during disease progression. Most studies of HIV-1 coreceptor utilization have relied on subtype B molecular clones, while a few have been conducted with primary isolates from other subtypes. Given the high prevalence of HIV-1 subtype C in the global epidemic and its predominance in the most heavily affected region of southern Africa, there is an obvious need to address whether viral genetic factors and/or a complex interaction between viral and host factors may be playing a yet unrecognized role in the spread of the virus.
It is worthy of note that the clone MJ4 replicated to significantly lower peak titers than the subtype B positive control in both PBMCs and macrophages. The reasons for this are not clear and may be just clone specific, especially because the positive control used in our experiments is a derivative of the laboratory-adapted molecular clone HXB2RU3, which is known to replicate to high titers. Further studies are needed to determine what factors may contribute to the differences observed in this study. Possible clone differences in entry mechanisms, transcription activity, packing capacity, induction of cytopathic effects, and other characteristics may individually or collectively play a role. However, it has been observed previously that HIV-1 subtype C primary isolates failed to replicate for as long as 28 days in macrophages until the macrophages were cocultured with Jurkattat
). The apparent failure of HIV-1 subtype C viruses to replicate to high titers may not be subtype specific but may simply imply that these viruses have the typical slow/low, non-syncytium-inducing (NSI) phenotype, like that found in some primary subtype B isolates (1
). The statistically significant differences observed in this study suggest that viral genetic factors, independent of cell donor differences, are responsible for the replication kinetics observed. We did not quantify the ability of MJ4 to induce cytopathic effects in PBMCs and macrophages, which may impact the ability of the virus to replicate to high titers in vitro. Recently, Chen et al. (8
) reported the construction of chimeric simian/human immunodeficiency viruses (SHIV) that bear HIV-1 subtype C envelope glycoproteins. Although these envelope glycoproteins were functional, some of the constructs failed to replicate in vitro but were able to infect experimental primates. The authors suggested the possibility that the target cell in vivo for subtype C envelope-bearing SHIV may be missing in PBMC culture. Taken together, these studies warrant further investigation into HIV-1 subtype C biology.
The presence of extra NF-κB enhancer element sites within subtype C LTRs has led to the speculation that these viruses may have a replicative advantage in target cells (4
). This argument is bolstered by transfection experiments involving reporter gene assays that show higher transcriptional activation of HIV-1 subtype C LTRs compared to that for other subtypes. However, few studies have compared the replication or cytopathic effects of viral isolates or clones of different subtypes in diverse primary target cells. It is noteworthy that these studies have not exhaustively explored the contribution of biologically relevant cofactors, such as proinflammatory cytokines, to the replication of HIV-1 subtype C in laboratory experiments. In this study, we compared the replication kinetics of two different clones belonging to subtypes B and C. We have shown that HXB2RU3CI (subtype B) replicates to higher titers than MJ4 (subtype C) in macrophages and PBMCs. Future studies will include the investigation of how factors such as cytokines may influence HIV-1 replication in a subtype-dependent manner, if at all.
The epidemic spread and pathogenesis of HIV-1 subtype C may differ from those of other subtypes. Studies in Tanzania show that HIV-1 subtype C has a higher odds ratio of perinatal transmission than the other two subtypes (38
). In circulating intersubtype recombinants, certain genetic loci of subtype C appear to have been selected in association with transmission (5
). In Kenya, a cross-sectional study showed that plasma HIV RNA levels were highest, and CD4 lymphocyte counts were lowest, among women infected with HIV-1 subtype C compared to those infected with subtypes A and D (32
). In a west African cohort of female commercial sex workers, individuals infected with subtype C were reported to be more likely to develop AIDS than those infected with subtype A (23
This is the second infectious molecular clone of HIV-1 subtype C reported, the first having been cloned from an Indian isolate after propagation in immortalized cell lines (26
). HIV-1 vaccine development, targeted mainly at rapidly spreading strains, is a priority. This clone and others like it are more representative of these genotypes and will be important tools for identifying correlates of protection, epitopes that may confer resistance to infection or disease, as well as for facilitating the testing of potential vaccines.