Background:  Studies in Zimbabwe and South Africa of HIV-1 subtype C viruses have identified CXCR4-tropic and syncytium-inducing viruses with increasing frequency, and evolution of drug resistance mutations in RT and protease genes under drug pressure. Here we examine env coreceptor usage in patients failing antiretroviral therapy (ARV) after 2 years.

Methods: In 2001 and 2003 virus isolates were collected from 11 subtype C-infected, treated Zimbabwean patients. Viral load (Roche Amplicor 1.5), CD4 counts (FACS), and syncytium-inducing/non-syncytium-inducing phenotypes (identified by co-culture with MT2 cells) were determined. In syncytium-inducing viruses, isolates and limiting dilution biologic clones were subjected to env sequencing and tropism assays with X4 and R5 transfected GHOST cells. Phenotype was predicted from sequence for 4 samples without viral isolates according to an increase in net positive charge and distinct amino acid substitutions in the V3 loop. Genetic distances between subsequent isolates were calculated with Dnadist within Phylip (Kimura-2 parameter).

Results:  In 3/11 patients with a decrease in viral load from a mean of 4.09 in 2001 to <2.6 log₁₀ copies/mL in 2003, CD4 counts increased from a mean of 56 in 2001 to 258 in 2003. In 8/11 patients with an increase in viral load from a mean of 3.93 to 4.83 log₁₀ copies/mL, CD4 counts decreased from a median of 114 to 21, and 1 to 6 RT resistance mutations accumulated. Of these 8 failing patients, 3 had non-syncytium-inducing virus both in 2001 and 2003, with no change in CD4 counts, while 4 that had syncytium-inducing virus in 2001, had a loss of CD4 cells, from a median of 142 to 16 CD4 cells/mL (p = 0.06; Wilcoxon rank test). Syncytium-inducing virus persisted in 3 of these 4. A fifth patient with persistent syncytium-inducing virus had an increase of 3 to169 CD4 cells/mL. Analysis of 26 clones of the 7 syncytium-inducing 2001 isolates with 2 to 5 LDC per isolate identified 2 isolates with only dual-tropic clones, 2 with X4 and 3 with dual tropic/X4 mixtures, with no R5 clones. Despite high replication rates during these 2 years, there was no difference in mean genetic distance between syncytium-inducing and non-syncytium-inducing consensus env sequences (p = 0.54, Student’s t test).

Conclusions:   Subtype C HIV-1 syncytium-inducing phenotype is associated with disease progression, evolution of drug resistance mutations and a steep decline in CD4 cells, as in subtype B. Subtype C syncytium-inducing biologic clones demonstrate dual tropism, in which cloned viruses replicated in both R5 and X4 cell lines, mixtures of distinct X4 and X4R5 dual tropism and in some cases a predominant X4 tropism.

Keywords: subtype C; envelope; coreceptor tropism