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266 Characterisation and Phylogenetic Analysis of the Complete env and gag Genes for HIV-1 Subtype C Isolates from South Africa
T. de Villiers*1, C. Sampson1, J. zur Megede2, S. Barnett2, S. Englebrecht1, and E. Janse van Rensburg1
1Univ. of Stellenbosch, Tygerberg, South Africa and 2Chiron Corp., Emeryville, CA, USA
Background: The Env proteins of HIV-1 are primary targets for neutralising antibodies, and gag-specific CTL responses are important in controlling viral load. These genes are therefore prime targets for vaccine research. Only 1 full-length env and gag gene sequence from South Africa has been characterised and published. We characterised the env and gag genes of 13 South African HIV-1 subtype C primary isolates.
Methods: PCR fragments containing the env and gag genes were cloned and sequenced. Sequence assembly, multiple sequence alignments, predicted amino acid translations, and phylogenetic analysis were performed with CLUSTALX, GENEDOC, and TREECONW software packages, respectively.
Results: Phylogenetic analysis of env and gag sequences revealed a distribution across several subtype C subclusters. The diversity among our isolates was comparable to the diversity seen amongst the C subtype as a whole. The V3 loop region in env was relatively conserved in all of our strains when compared with other subtypes, but the region immediately downstream was highly variable. A highly conserved N-linked glycosylation site proximal to the first cysteine residue, observed in all HIV-1 subtypes except subtype C, was also absent in the V3 region of our strains. The tetrapeptide crown sequence of the loop was GPGQ for all our C subtypes. An NSI phenotype conferred by uncharged or negatively charged amino acid residues at specific positions in the V3 loop was subsequently confirmed in cell culture experiments. A variation of 6(13% was observed in subtype C gag sequences. The highest variation within gag was seen in the p17 matrix protein (8(25%).
Conclusions: Our isolates were distributed across several subtype C subclusters with a high diversity, indicating multiple independent introductions of strains of this subtype into Southern Africa. The molecular characterisation of our functional env and gag genes derived from primary South African HIV-1 isolates will enable us to select candidate subtype C antigens for further vaccine development.
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