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267 Biological Characterization of South African HIV-1 Subtype B and C Isolates
F. Treurnicht*, T.-L. Smith, S. Engelbrecht, M. Claassen, B. Robson, M. Zeier, and E. Janse van Rensburg
Tygerberg Hosp. and Univ. of Stellenbosch, South Africa
Background: CCR5 was identified as the principal coreceptor needed for entry by primary macrophage-tropic, nonsyncytium-inducing (NSI) HIV-1 strains into CD4+ target cells. T-cell-tropic, syncytium-inducing (SI) HIV-1 isolates use the CXCR4 coreceptor for viral entry. Subtype C strains from Ethiopia were reported to cause cytopathic effects in primary cultures, but were NSI in MT-2 cells and used CCR5 as principal coreceptor.
Methods: HIV-1 was isolated in 1998(1999 from 18 primary cultures by cocultivation with PHA-stimulated HIV-1-negative donor peripheral blood mononuclear cells. SI or NSI phenotype was determined in MT-2 cells. Coreceptor usage of the primary isolates was determined in HOS-CD4 cell lines expressing different coreceptors: CCR1, CCR2B, CCR3, CCR4, CCR5, and CXCR4. The HIV-1 subtype was determined by V3 competitive enzyme immunoassay (cPEIA). The serotyping results were confirmed by PCR and sequencing of an env gene fragment that includes the V3 loop from plasma RNA and DNA prepared from infected cultures. Multiple sequence alignment, predicted amino acid translations, and phylogenetic analysis were performed with the CLUSTALX, GENEDOC, and TREECONW software packages, respectively.
Results: All the isolates were NSI in both the primary cultures and the MT-2 cells. The amino acid charge of the V3 loop of the viral strains correlated with their NSI phenotypes. The V3 cPEIA and phylogenetic analysis of the partial gp120 region gave concordant results on the 15 subtype C strains. The 3 B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides. Sixteen of the isolates used only CCR5 as coreceptor, whereas 2 isolates, 1 subtype C and 1 subtype B, made use of additional coreceptors including CXCR4. Two patients' viruses remained mainly CCR5-tropic and NSI despite of having absolute CD4+ cell counts less than 100.
Conclusions: All our subtype C isolates are phenotypically NSI strains and use CCR5 as principal coreceptor. Although 1 B isolate showed the ability to utilize coreceptors other than CCR5, all 3 of them replicated the best in CCR5 expressing cells.
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