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. 2003 Jun;77(12):6811-22.
doi: 10.1128/jvi.77.12.6811-6822.2003.

Turnover of env variable region 1 and 2 genotypes in subjects with late-stage human immunodeficiency virus type 1 infection

Kathryn M Kitrinos  1 , Noah G Hoffman, Julie A E Nelson, Ronald Swanstrom
Affiliations

Turnover of env variable region 1 and 2 genotypes in subjects with late-stage human immunodeficiency virus type 1 infection

Kathryn M Kitrinos et al. J Virol. 2003 Jun.

Abstract

The env gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of env (V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their env variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.

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Figures

FIG. 1.
FIG. 1.
V1/V2-HTA analysis using the four probes developed from the molecular clones Ba-L, JR-FL, NL4-3, and YU-2. The probes were annealed with PCR products of all four probes. The samples are identified by a letter indicating which PCR product was used in each lane: B, Ba-L; J, JR-FL; N, NL4-3; Y, YU-2. The position of the radiolabeled, single-stranded probe is indicated by an arrow.
FIG. 2.
FIG. 2.
V1/V2-HTA analysis of entry and ending blood plasma samples. V1/V2-HTAs were performed on first and last samples with both the Ba-L and JR-FL probes; the HTAs with the Ba-L probe are shown for 1025, 1036, 1052, 1053, 1073, 1103, 1116, 1124, 1133, and 1146, while the HTAs with the JR-FL probe are shown for 1004, 1007, 1012, 1018, 1024, 1027, 1063, 1066, 1067, 1079, and 1114. Samples are identified by number of days from the start of the trial. The position of the radiolabeled, single-stranded probe is indicated by an arrow, and the probe homoduplexes are not shown. One subject (1025) had a heteroduplex band that migrated above the single-stranded probe, and one subject (1124) had a heteroduplex band that migrated close to the single-stranded probe. The bands above the single-stranded probe in other subjects (including 1004 and 1146) are additional single-stranded probe bands differentially bound to the PCR primer.
FIG.3.
FIG.3.
Longitudinal analysis of V1/V2 genotypic variants. V1/V2-HTAs were performed on all monthly samples from eight subjects and shown with either the Ba-L probe (1036, 1053, 1073, 1124, and 1133) or the JR-FL probe (1027, 1066, and 1079). The samples are identified by number of days from the start of the trial (above each lane). Only the heteroduplex region of each gel is shown. The predominant transition period for each subject is indicated by a bracket. SS, single-stranded probe. The small numbers on the sides of the gels are identifiers for the genotypic variants for which sequence information was obtained. Cartoon depictions of the V1 and V2 regions are shown for each sequenced variant. Lines represent nucleotide sequence differences, and dots represent deletions relative to the other variants. Half-lines are used to show the positions of minor sequence variants. These minor variants were linked when displayed attached to the upper line. Each cartoon represents the consensus sequence for each variant. The number to the left of the cartoon corresponds to the identifying number on the gel, and the number to the right indicates the number of clones obtained and sequenced for each variant. The absence of a number to the right of the cartoon indicates that the PCR product was bulk sequenced. An asterisk to the right of the cartoon indicates that the variant has sequence differences in the C2 region, which are not shown.
FIG. 4.
FIG. 4.
V1/V2 variants analyzed based on the absence or presence of length differences. The graph displays pairwise comparisons of the V1/V2 variants present at entry and the newly emerged V1/V2 variants, broken down into variants with and without size differences. The y axis displays the number of nucleotide differences for each variant. Circles, individual variants with a mutation ratio greater than or equal to 2:1 in either V1 or V2 (at least twice as many mutations in one region over the other); squares, individual variants with a mutation ratio less than 2:1 in either V1 or V2; triangles, individual variants with only one mutation across V1/V2.
FIG. 5.
FIG. 5.
Nucleotide differences and size differences in newly emerged V1/V2 variants. The nucleotide sequence of each newly emerged variant was compared to the nucleotide sequence of an entry variant that was the closest related variant. For each variant, a coding point mutation change is noted by an arrow where the change occurred. Numbers in parentheses next to the subject identification numbers are the total number of coding changes in the V1/V2 region for that variant. Numbers above or below the arrows indicate that more than one coding change occurred within one codon. Size differences are noted by an asterisk, with the change in the number of nucleotides noted above the asterisk.
FIG. 6.
FIG. 6.
Composition of asparagine (N), serine, (S), and threonine (T) codons in an alignment of 183 full-length gp120 coding sequences from the Los Alamos HIV sequence database. Each point in the plot represents the center of a 25-codon sliding window. The percentage of all aligned codons in each window that are AAT, ACT, or AGT is indicated by the gray-filled plot; the heavy black line shows the total percentage of all other asparagine, serine, or threonine codons. The sum of the two plots is therefore equal to the percentage of all asparagine, serine, or threonine codons. Vertical lines demarcate the regions of length polymorphism in V1, V2, V4, and V5 and indicate the positions of the cysteines flanking V3. The percentages of all asparagine, serine, or threonine codons of either category in each region of gp120 are shown at the top of the plot.
FIG. 7.
FIG. 7.
Example of recombination between V1 and V2 in vivo. The V1/V2-HTA time points from subject 1133 from before and after the recombination events are shown on the left. The cartoon depicts the time points shown in the gel. The V1/V2 variants involved in the recombination event are lettered similarly on the gel and in the cartoon. Major V1/V2 genotypic variants that were not involved in the recombination event are marked on the gel with an asterisk. Different V1 and V2 sequence motifs are distinguished by shading and hatching. SS, single-stranded probe. The V1/V2-HTA lanes are from Fig. 2.
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