Molecular characteristics of human immunodeficiency virus type 1 subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies

Abstract

The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or "lineages" of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.

FIG. 1.

Representative pol tree showing the relationships between retrospective and contemporary sequences from South Africa, Botswana, and other countries affected by the subtype C epidemic. The sequences are coded by the country of origin and year of isolation. The following sequences were included in the analysis: 49 previously described isolates from Botswana (accession numbers AF110960, AF110963, AF110967, AF110970, AF110972, AF110973, AF110978, and AF443074 to AF443115), 9 sequences from India (accession numbers AF286232, AF286223, AF286231, AB023804, AF067159, AF067155, AF067154, AF067157, and AF067158), 4 sequences from Tanzania (accession numbers AF286234, AF286235, AF361874, and AF361875), 2 sequences from Zambia (AF286224 and AF286225), 2 sequences from Brazil (U52853 and AF2862228), 1 sequence from Ethiopia (U46016), 1 sequence from Israel (AF286233), and 69 sequences from South Africa, including 5 previously described sequences (AF286227, AY043173, AY043174, AY043175, and AY043176), 3 sequences from another study (71), and 61 sequences newly generated from this study (14 retrospective and 47 contemporary strains).

FIG. 2.

Phylogenetic relationship of C2V5 envelope sequences from KwaZulu-Natal, Botswana, Zambia, and Tanzania. Non-KwaZulu-Natal strains are the same as those described in Fig. 1.

FIG.3.

Correlation of signature patterns with structure and function for protease and reverse transcriptase. conKZN, KwaZulu-Natal consensus; conA, conB, conC, and conD, consensus sequences for subtypes A, B, C, and D, respectively; APV, SQV, RTV, NFV, INV, drug binding sites for amprenavir, saquinavir, ritonavir, nelfinavir, and indinavir, respectively; functⁿ, RT, reverse transcriptase; CTL, cytotoxic T-lymphocyte epitope; •, drug-binding site; k, protein kinase C phosphorylation site; c, casein kinase phosphorylation site; m, myristoylation site; aaaa, amidation site; t, tyrosine kinase phosphorylation site; g, cyclic AMP- and cyclic GMP-dependent protein kinase site; T, thiocarboxanilide UC-781; N, nevirapine; Q, quinoxaline HBY 097; E, efivirenz; a, accessory mutation; P, primary mutation; caret, extended β-strand; S, bend; star, hydrogen-bonded turn; h, helix; p, purifying selection pressure; d, Darwinian (positive) selection pressure.

FIG. 4.

Correlation of signature patterns with structure and function of V3 loop. KNZenv, KwaZulu-Natal consensus; Con_A, Con_B, Con_C, and Con_D, consensus sequences for subtypes A, B, C, and D, respectively; k, protein kinase C phosphorylation site; c, casein kinase phosphorylation site; n, N-linked glycosylation site; caret, extended β-strand; h, helix; 4, CD4⁺ binding site; d, Darwinian (positive) selection pressure.