Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination

Abstract

The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.

FIG. 1

Plots of similarity (generated by SimPlot) of a set of reference sequences to the 93IN999 (upper panel) and 95IN21301 (lower panel) genomes. Each curve is a comparison between the genome being analyzed and a reference genome. Each point plotted is the percent identity within a sliding window 600 bp wide centered on the position plotted, with a step size between points of 20 bp. Positions containing gaps were excluded from the comparison. The horizontal bars above the curves are a cartoon of the open reading frames of the HIV-1 genome, arranged as indicated in Fig. 3. The colors are consistent with those used for the similarity curves and indicate the subtype to which that part of the genome is most similar based on the adjacent similarity plot. Results for the remaining four genomes discussed in this report were consistent with those for 93IN999.

FIG. 2

(A) Similarity plot as in Fig. 1 for the env gene of isolate 95IN21301, with a window size of 200 bp and a step size of 10 bp. The subtype reference sequences were majority (50%) consensus sequences for each of the subtypes, obtained from the Los Alamos web site (http://hiv-web.lanl.gov). The dashed regions indicate areas in V1 and V2 in which less than 50% of the sites could be compared due to gaps or lack of subtype consensus. Dotted vertical lines indicate breakpoints identified by maximization of χ² as described in Materials and Methods, with numbers of informative sites shared by 95IN21301 and the subtype in each bounded region indicated below in the color assigned to that subtype. P values were calculated by using Fisher’s exact test. Four-member trees consistent with these sites are shown at the left. Above are phylogenetic trees for each region bounded by the recombination breakpoints showing the proportion of 100 bootstrapped trees surrounding the indicated relationship. The predicted gp120/gp41 processing site is at base 2044 in this alignment. (B) Similarity plot as in panel A for the nef gene and the U3/R LTR region of isolate 95IN21301. The LTR begins at position 296 in this alignment, and the nef termination codon is at position 634. Subtype majority consensus sequences were determined by using SF170, U455, and UG037 (subtype A), RF, MN, and TH475 (subtype B), and C2220, BR025, 93IN904, 93IN905, 93IN999, 94IN11246, and 95IN21068 (subtype C).

FIG. 3

Cartoon depicting the subtype assignment of each region of the HIV-1 genome for all characterized A/C recombinant genomes.

FIG. 4

Phylogenetic tree based on the env gene sequences compared in the HMA reaction used to identify genotypes. The tree is based on 834 sites that remained after gap stripping of the alignment predicted for the ED5 to ED12 PCR product. Numbers at nodes indicate clades supported in more than 50 of 100 bootstrapped trees, and the scale for genetic distance is indicated below. The prototype sequences for the subtypes indicated were as follows: A1, RW020; A3, SF170; C1, MA959; C2, ZAM18; C3, IN868; and C4, BR025.

FIG. 5

Phylogenetic tree comparing the sequences reported here to Indian sequences reported previously for pol (692 bp) (54), with bootstrap values greater than 50% indicated. The representatives for each of the subtypes were as follows: A, U455; B, RF; C, C2220 and BR025; D, NDK; and G, 92NG083.

FIG. 6

Distributions of amino acid sequence differences in epitopes (A) and overall gene sequences (B). All differences in sequences are shown on the left, while only nonconservative differences identified based on physicochemical properties are shown on the right.