Structural Features and Genetic Diversity in Gag Gene of Rare HIV-1 Subtypes from the Democratic Republic of Congo

Type-1 HIV (HIV-1) group M (HIV-1M) genetic diversity is highest in the Congo Basin where the epidemic ignited a century ago. HIV-1M has diversified into multiple subtypes, sub-subtypes, and circulating and unique recombinant forms (CRFs/URFs). An unanswered question is why some rare subtypes never reached epidemic levels despite their age. Several studies identified the role of HIV-1M accessory genes nef and vpu in virus adaptation to human hosts and subsequent spread. Other reports also pointed out the pivotal role of gag in transmissibility, virulence, and replication capacity. In this study we characterized the HIV-1 gag gene of 148 samples collected in different localities of the Democratic Republic of the Congo (DRC) between 1997 and 2013. We used nested polymerase chain reaction (PCR) to amplify the whole gag gene. PCR products were sequenced either by Sanger method or by next generation sequencing on Illumina MiSeq or iSeq100 platforms. Generated sequences were used for subsequent analyses using different bioinformatic tools. Phylogenetic analysis of the generated sequences revealed a high genetic diversity with up to 22 different subtypes, sub-subtypes, CRFs. Up to 15% (22/148) URFs were identified, in addition to rare subtypes such as H, J, and K. At least two amino acid motifs present in the gag gene have been shown to modulate HIV-1 replication, budding, and fitness: the P(T/S)AP and the LYPXnL motifs. Structural analysis revealed the presence of P(T/S)AP in all the 148 sequences with the majority (136/148) bearing the PTAP. Three samples presented a duplication of this motif. The LYPXnL motif was identified in 38 of 148 sequences. There was no clear link between the frequency of these motifs and HIV-1M subtypes. In summary, we confirmed a high genetic diversity of HIV-1M in the DRC. We observed the presence of amino acid motifs important for viral replication and budding even in some rare HIV-1 subtypes. Their impact on viral fitness needs be further evaluated by in vitro studies.