Characterization of V3 sequence heterogeneity in subtype C human immunodeficiency virus type 1 isolates from Malawi: underrepresentation of X4 variants

Abstract

We have examined the nature of V3 sequence variability among subtype C human immunodeficiency virus type 1 (HIV-1) sequences from plasma-derived viral RNA present in infected men from Malawi. Sequence variability was assessed by direct sequence analysis of the V3 reverse transcription-PCR products, examination of virus populations by a subtype C V3-specific heteroduplex tracking assay (V3-HTA), and selected sequence analysis of molecular clones derived from the PCR products. Sequence variability in V3 among the subtype C viruses was not associated with the presence of basic amino acid substitutions. This observation is in contrast to that for subtype B HIV-1, where sequence variability is associated with such substitutions, and these substitutions are determinants of altered coreceptor usage. Evolutionary variants in subtype C V3 sequences, as defined by the V3-HTA, were not correlated with the CD4 level in the infected person, while such a correlation was found with subtype B V3 sequences. Viruses were isolated from a subset of the subjects; all isolates used CCR5 and not CXCR4 as a coreceptor, and none was able to grow in MT-2 cells, a hallmark of the syncytium-inducing phenotype that is correlated with CXCR4 usage. The overall sequence variability of the subtype C V3 region was no greater than that of the conserved regions of gp120. This limited sequence variability was also a feature of subtype B V3 sequences that do not carry the basic amino acid substitutions associated with altered coreceptor usage. Our results indicate that altered coreceptor usage is rare in subtype C HIV-1 isolates in sub-Saharan Africa and that sequence variability is not a feature of the V3 region of env in the absence of altered coreceptor usage.

FIG. 1

Examples of V3-HTA patterns obtained with the subtype C probe. RT-PCR products were generated from viral RNA isolated from the plasma of subjects infected with HIV-1 subtype C. Lanes: 1, probe without PCR product; 2, probe with PCR product generated from the D516-11 probe plasmid; 3 to 18, examples of RT-PCR products from subjects with either a single dominant heteroduplex band or multiple heteroduplex bands. Starting with lane 3, the order is as follows: S115, C011, S081, C044, S071, S200, S021, C065, S180, C111, C034, S073, S031, S123, S134, and S191. The positions in the gel of the single-stranded probe (A), probe homoduplex (B), and heteroduplexes with the most rapid migration (C) are shown.

FIG. 2

Alignment of inferred V3 amino acid sequences corresponding to V3-HTA bands with mobility ratios of less than 0.85. Each sequence represents a single clone from the RT-PCR product from that subject. The mobility of each clone was verified by V3-HTA. Basic amino acid substitutions at SI-associated positions are underlined; deletions are designated by Δ.

FIG. 3

V3-HTA mobility ratios plotted against the CD4⁺-T-cell count. Only the lowest mobility ratio was used for samples with multiple bands. The data for the subtype C viruses were taken from Table 1. The data from the subtype B viruses were generated from samples described in Materials and Methods.

FIG. 4

Sequence variability of subtype C V3 sequences compared to the variability of NSI/R5-like and SI/X4-like subtype B V3 sequences. The consensus sequence for subtype C was generated from a data set of 121 sequences. The NSI/R5-like and SI/X4-like variability patterns are from reference (56). Substitution percentages were calculated by dividing the sum of the substitutions away from the consensus by the total number of amino acids.

FIG. 5

Evolutionary distances of segments of gp120. Groups of sequences of the gp120 coding region for subtype C, subtype B R5-like, and subtype B X4-like viruses were assembled from the Los Alamos HIV-1 Sequence Database. A description of the sequences used is available on request. The sequences were aligned and then divided into segments of between 26 and 50 codons. These segments are indicated in the figure below the line, using the HIV-1_JR-FL numbering. The region in C3 just downstream of V3 was omitted because of its recently described position as an external loop (loop E) in the protein structure with associated sequence variability (49). Evolutionary distance was calculated for each segment for each group of sequences. The distance of each segment within a group was compared to the equivalent distance in the other two groups to determine if they were significantly different. The two cases where a statistically significant difference was observed are noted with an asterisk. The vertical thin lines show standard error.