A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand , in fulfillment of the requirements for the degree of Master of Science in Medicine, 2013 Sequence analysis from HIV-1 (human immunodeficiency virus type 1) subtype B and more; recently subtype C infected patients has revealed that mutations in the HIV-1 protease region; that confer drug resistance to boosted protease inhibitor (PIs) are rarely detected at the time; of virological failure. Mutations in the HIV-1 subtype B gag-pol cleavage sites are thought to; be compensatory mutations which arise as a result of PI use. This study investigated the; presence of compensatory mutations in the HIV-1 subtype C gag-pol cleavage sites and; matched pol genotypes from South African patients failing a boosted PI-based regimen, as; compared to antiretroviral drug naïve patients.; A new amplification protocol encompassing the near full-length gag, PR and partial RT was; established and used to sequence the HIV-1 gag-pol cleavage sites from 23 proviral DNA; samples (p24 antigen cultured peripheral blood mononuclear cells; PBMCs), and 51 patient; samples (23 antiretroviral drug-naïve, 26 failing second-line lopinavir/ritonavir containing; regimens), all attending the Charlotte Maxeke Johannesburg Hospital. Nucleotide sequences; were aligned and codon positions S373Q, A431V, I437T/V, L449P or P453L associated with; known gag-pol cleavage site mutations were analysed and compared. The pol genotypes were; established using an in house assay. Antiretroviral drug resistant primary virus isolates were; grown from samples from patients enrolled on the CIPRA-SA study, and propagated in coculture; with PHA-activated, IL-2 stimulated PBMCs. HIV-1 gag-pol cleavage sites and pol; genotypes for all primary virus isolates were established as described above.; Fifty one of 74 patient samples, used to establish the in-house gag-pol cleavage site assay,; were successfully amplified and sequenced. Detailed analysis of the five known gag-pol; cleavage sites revealed that 5 patient samples (4 PI-exposed, 1 unknown regimen) encoded; for the previously described mutations that impact on gag-pol cleavage in the absence of any; major PR mutations. A further five samples from patients on the failing PI-based regimen had; major PR mutations. No known mutations in the gag-pol region were identified in patients; failing a first line regimen. The pol mutations described in this study were similar to the; findings reported for treatment failures in South African HIV-1 subtype C infected patients.; Primary virus was grown from only 25 of the 91 PBMC CIPRA samples. None of the 25; CIPRA-SA primary virus isolates had gag-pol cleavage site mutations, and only 9 harboured; known RT antiretroviral drug resistant mutations.; Overall, the presence of HIV-1 gag-pol cleavage site mutations may account for virological; treatment failure in 5 of the South African patient samples analysed. Although the gag-pol; cleavage site mutations detected in the current study are only present in a small proportion of; treatment-experienced South African patients, this may increase due to more patients; accessing second line PI-containing regimens. Thus, future genotyping work incorporating; the analysis of the gag-pol cleavage sites in addition to the PR and RT regions is warranted.; The antiretroviral drug resistant primary viruses obtained provide valuable reagents for future; phenotyping studies.