Elucidation of HIV-1 subtype C genetic diversity in gag and tat from the clinical cohorts of India: the PTAP motif duplication in p6 gag confers replication fitness on subtype C

Abstract

In the present study, using a prospective follow-up of two Indian clinical cohorts and fine dissection of the viral quasispecies in a subset of subjects, we demonstrate for the first time that subtype C is endowed with a unique ability to duplicate longer stretches of amino acid residues in p6 Gag. We show further that this ability to duplicate longer stretches of amino acid residues confers replication advantage on the variant viral strains in the natural infection and experimental conditions. The viral strains of HIV respond to environmental stresses, such as the immune response or antiretroviral therapy (ART), by causing insertions or deletion of sequences or by introducing appropriate amino acid substitutions, to acquire resistance against the restriction pressures. While the nature of the selection forces causing a specific type of sequence variations is not known in many cases, there appear to be a few hotspots within the viral genome where especially sequence insertions are more common under certain conditions. A location in the viral promoter between the RBEIII and NF-κB transcription factor binding sites offers an appropriate example. Further, the PTAP motif in the p6 protein of Gag provides a second example for sequence insertion hot-spot in response to drug pressure and pressures of unknown nature. While the mutation response is a universal phenomenon evident in all the genetic subtypes of HIV-1, subtype C appears to differ from the other viral subtypes in two important respects in this regard – the frequency of the sequence insertions and the length of the inserted sequences. First, in a cross-sectional analysis of a clinical cohort of Brazil, Martins et al. demonstrated the prevalence of sequence insertions in Gag to be highly superior in subtype C infection as compared to the infections of subtypes B and F (Martins et al., 2011). Importantly, the prevalence of the sequence insertions in Gag was as high as 25% in drug-naïve subjects of subtype C, which increased further to a staggering 52% in the ART-exposed subjects of this cohort. Second, in the present work, we demonstrate that unlike in non-C subtypes, subtype C viral strains demonstrate a potential to insert motifs of longer sequence length in p6 Gag in drug-naïve subjects of Indian cohorts. The ability to insert longer sequence motifs offers a great advantage to subtype C that the viral strains can duplicate sequence motifs of biological significance. We previously demonstrated that in the viral promoter, while all the HIV-1 subtypes are capable of duplicating the RBEIII binding site, only subtype C viral strains demonstrate a potential to duplicate the NF-κB binding site (Bachu M et al., ARHR). We demonstrated further that the emerging viral strains of subtype C containing four NF-κB binding sites in the viral enhancer have been expanding at a rapid rate in India replacing the canonical subtype C viruses that contain only three NF-κB binding sites (Bachu et al., 2012b). Thus, subtype C appears to exploit the phenomenon of sequence insertion as a powerful strategy to duplicate sequence motifs of biological significance to gain replication advantage. In the present work, we demonstrate for the first time that 14 amino acid sequence duplication in p6 Gag of subtype C leads to a near complete domination of the variant viral strains in six different subjects in the plasma viral RNA and the proviral DNA compartment. Using a range of viral assays and paired infectious molecular clones, we show an enhanced interaction between subtype C Gag and Tsg101, a host factor that is an integral component of the cellular endosomal sorting machinery (ESCRT), underlying the domination of the variant viral strains over the standard viral strains.