Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.

FIG. 1.

Relationship between viral load and estimated time since infection. Log₁₀ viral load (copies/milliliter) for the sample analyzed is plotted versus the estimated time after infection, assuming infection was 2 weeks prior to the onset of symptoms of acute infection. A vertical line is included at week 7 to indicate groups of subjects staged to before or after this time. Filled symbols are subjects whose virus showed a single V1/V2 variant (as assessed by HTA); open symbols are subjects whose virus showed multiple V1/V2 variants.

FIG. 2.

V1/V2-HTA patterns of subjects during primary HIV-1 infection. (A) Control lanes with the probes for V1/V2 diversity analysis (JR-FL and Ba-L). Probes were run alone with or without the denaturing step or with their own or alternate probe's PCR product. The asterisks indicate the single-stranded probe, and the lower bands are the probe homoduplex. (B) Blood plasma samples were used as the source of viral RNA which was then used as a template to amplify the V1/V2 region of env using PCR. Heteroduplexes were formed with either the JRFL or BAL V1/V2 probe, which were then resolved in a polyacrylamide gel. The identifier of each subject is shown above the lane. The band with the asterisk represents the position of the single-stranded HTA probe. The weeks postinfection (WPI) and the numbers of V1/V2 variants detected (#) are indicated at the bottom of each lane.

FIG. 3.

Comparison of number of V1/V2 variants detected by HTA with time after infection. Each datum point represents one subject from the primary infection cohort. The estimated time postinfection is plotted versus the number of V1/V2 variants detected for the subject (Fig. 2). Subjects in the primary infection cohort are compared to a separate group of subjects during the chronic stage of their HIV-1 infection. Although not resolved in the figure, there are nine individuals identified with single variants prior to week 7.

FIG. 4.

Phylogenetic analysis of V1/V2 clones. RT-PCR products from separate reactions independently cloned after gel purification. Variant clones were identified by HTA screening. Where possible, at least two independent clones of each variant were sequenced. For two subjects, Z06 and Z24, the single variant was independently cloned and sequenced 6 to 10 times from each RT-PCR product. The neighbor-joining tree was made with aligned sequences with gaps ignored. All sequences are from the blood. Identifiers indicate subject (e.g., Z10), clone (e.g., g3), and HTA band (e.g., 0.1, 0.2, etc.), respectively. For subjects Z06 and Z24, which each had a single variant, the HTA band number is indicated as “0.” For these two subjects, the number of clones with identical sequences is shown in parentheses.

FIG. 5.

V3-HTA patterns of subjects during primary HIV-1 infection. (A) Control lanes with the clade B (JN27) and clade C (JN33) V3 probes alone or against their PCR products. The single-stranded probe is indicated by the top arrows, and the lower arrow indicates the probe homoduplex. (B) Viral RNA from blood plasma was used as a template to amplify the V3 region of env using PCR. Heteroduplexes were formed between the PCR product and either the pJN27 (clade B) or pJN33 (clade C) V3 HTA probes. The resulting heteroduplexes were resolved in a polyacrylamide gel. Subject identifiers are shown at the bottom of the each lane. WPI, weeks postinfection; #, number of V3 variants detected.

FIG. 6.

Phylogenetic analysis of plasma gag sequences. The sequence from the bulk PCR product covering two overlapping regions of gag p17 and p24 were combined for this analysis. Subject numbers are indicated. Branch numbers represent number of nucleotide changes per 100 bases.

FIG. 7.

Comparison of V1/V2 env variants in blood and semen of subjects during primary HIV-1 infection. Viral RNA extracted from paired blood plasma (B) or SP (S) was used as a template to amplify the V1/V2 region of env using PCR. Subject identifiers are above the lane. Heteroduplexes were formed and analyzed as described in the legend of Fig. 2.

FIG. 8.

Comparison of V1/V2 env variants in blood and CSF of subjects during primary HIV-1 infection. Viral RNA extracted from paired blood plasma (B) or CSF (C) was used as a template to amplify the V1/V2 region of env using PCR. Subject identifiers are above the lane. Heteroduplexes were formed and analyzed as described in the legend of Fig. 2.

FIG. 9.

Genetic distances of sequences from viruses present in the primary infection cohort to a large set of HIV-1 sequences. Regions of the gag (A) and env (B) genes were amplified by RT-PCR, and the purified PCR products were sequenced by using one of the PCR primers. The amino acid sequence was inferred from the nucleotide sequences. Pairwise comparisons were made for percent amino acid identity between each sequence and the clade B consensus sequence (○) and to a large set of HIV-1 sequences. For the large comparison, the mean value (+), the inner quartile range (box), and the entire range (line) are shown.