Reverse Transcriptase Connection and RNase H Domain Variation in HIV-1 Subtype C Among Individuals with Virologic Failure in Botswana

Background: Classical reverse transcriptase (RT) drug resistance mutations (DRMs) are primary determinants of antiretroviral treatment failure, but polymorphisms within the RT connection and RNase H domains may also influence RT inhibitor susceptibility. These regions remain poorly characterised in HIV-1 subtype C (HIV-1C), the predominant subtype in sub-Saharan Africa. This study investigated RT connection and RNase H polymorphisms in treatment-experienced people with HIV (PWH) experiencing virologic failure (VF) in Botswana. Methods: Seventeen plasma samples from the Botswana National HIV Drug Resistance Programme with documented VF (viral load >200 copies/mL) and prior Sanger resistance results were selected. One sample was excluded because of insufficient Oxford Nanopore Technologies (ONT) sequencing coverage, leaving 16 samples for analysis. Samples were amplified using the DeepChek® HIV-1 Full PR/RT/INT assay and analysed using the HIVgenomeR™ v2.0 ONT pol DRM pipeline. ONT-derived RT resistance profiles were compared with historical Sanger results, and RT connection and RNase H polymorphisms were characterised. Results: ONT identified additional RT DRMs in five sequences previously classified as lacking classical RT DRMs. Of the remaining six sequences without classical DRMs, four harboured minority RT variants. RT connection polymorphisms were common, including G335D (13/16) and T377M (9/16). Recurrent mutation combinations included T377M–T470A, A371V–E399D, T377M–E399D, and T377M–A360T. Conclusions: The extended HIV-1 pol genotyping assay enabled characterisation of the RT connection and RNase H domains while improving detection of classical RT DRMs and minority variants. These findings support further investigation of extended RT sequencing for HIV-1C molecular surveillance.