Characterisation of RT Connection and RNase H Polymorphisms in HIV-1 Subtype C in Botswana

Emerging evidence suggests that polymorphisms in the reverse transcriptase connection (RT-conn) and RNase H domains may contribute to resistance to reverse transcriptase inhibitors (RTIs). Here, we characterised the polymorphic landscape of the RT-conn and RNase H domains in HIV-1 subtype C (HIV-1C) from Botswana across the pre-ART and post-ART eras, including treatment-naïve (TN) and treatment-experienced (TE) individuals. A total of 1571 HIV-1C sequences were analysed: 76 pre-ART (≤2002) and 1495 post-ART (>2002) sequences were obtained from the Los Alamos database and the Botswana Combination Prevention Project (2013-2018). Post-ART sequences were stratified into TN (n = 1282) and TE individuals with virologic failure (TEVF, n = 213). Naturally occurring and ART-associated polymorphisms within RT-conn (aa 321-440) and RNase H (aa 441-560) were assessed. Among TN individuals, 12 polymorphisms exceeded 5% pre-ART, including R461K and L491P, while 31 polymorphisms were observed post-ART, indicating a temporal shift. Several substitutions were significantly higher in TEVF and showed a history of thymidine analogue-, tenofovir- and lamivudine/emtricitabine-based exposure. Covariant analysis identified significant co-occurrence of polymerase mutations (M184V/I, D67N) with RT-conn/RNase H substitutions (p < 0.05). These findings demonstrate HIV-1C evolution within the extended RT domains under ART pressure and support their inclusion in molecular surveillance frameworks in Botswana.