Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

Abstract

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

Fig 1. HIV-1 Full-length genome phylogenetic analysis of six epidemiologically-linked heterosexual transmission pairs.

(A) Nucleotide sequences for all 115 single genomes amplified from six linked transmission pairs were aligned to the curated LANL consensus/ancestral alignment and a maximum likelihood tree was generated. (B) Single genome nucleotide sequences for each viral gene (gag, pol, vif, vpr, vpu, tat, rev, env, & nef) were translated to their amino acids and then concatenated. These were aligned with LANL consensus/ancestral concatenated protein sequences and a maximum likelihood tree was generated. Transmitted/founder sequences from linked recipients are in blue, donor non-transmitted variants are in red, LANL database curated consensus/ancestral sequences are shown in black, where the LANL subtype C consensus is indicated by a green circle. Black arrows indicate virus variants from the donor quasispecies that were selected for generation of non-transmitted (NT) infectious molecular clones.

Fig 2. Transmission selects for more consensus-like TF variants.

The pairwise distance of each viral variant on the (A) nucleotide and (B) amino acid phylogenetic trees to the LANL subtype C consensus node were measured and plotted for each transmission pair. Transmitted/founder variants are in blue, and non-transmitted variants are in red. The median of the non-transmitted variants is designated with a black line. The statistical significance of the difference between TF and NT donor median values was analyzed using a one-tailed Wilcoxon matched-pairs signed rank test.

Fig 3. Particle infectivity of TF and NT infectious molecular clones.

293T cells were transfected with TF (blue) and NT (red) infectious molecular clones. Supernatants collected 48 hours post-transfection were titered on TZM-bl cells to define the number infectious units per microliter (IU/ul), while reverse transcriptase activity was measured simultaneously for total viral particles per microliter (RT DLU, reverse transcriptase digital light units). The particle infectivity (IU/RT DLU) of each infectious molecular clone is plotted for each transmission pair. The median of the NT variants is designated with a black line. The statistical significance of the difference between TF and NT donor median values was determined using a two-tailed Wilcoxon matched-pairs signed rank test (p = 0.6875).

Fig 4. TF variants are more sensitive to neutralization by donor plasma than NT viruses.

(A) Neutralization of TF (blue) and NT (red) IMC by donor plasma was measured for each pair in a TZM-bl neutralization assay. Percent neutralization by donor plasma (diluted 1:100) is depicted on the y-axis, and representative TF and NT viruses tested for each transmission pair are depicted on the x-axis. The median of the NT variants is designated with a black line. The statistical significance of the difference between TF and NT donor median values was determined using a two-tailed Wilcoxon matched-pairs signed rank test (p = 0.031). (B) Spearman correlation of the pairwise distance to the amino acid subtype C consensus and donor plasma neutralization described in part A over all the variants tested from 6 transmission pairs (p = 0.011, r = -0.4995).

Fig 5. In vitro replication of TF and NT viruses in PBMC.

(A) Virus growth over 10 days in PBMC culture as measured by reverse transcriptase (RT) activity (DLU = digital light units) of the TF (blue), NT variants (red) and MJ4 standard (black) for one representative transmission pair, 331. (B) Replicative capacity (RC) scores, based on the area under the curve relative to MJ4, of all tested TF (blue) and NT (red) variants from six transmission pairs. (C) RC scores of the TF (blue) compared to the median of the corresponding NT variants (red) (Wilcoxon matched-pairs signed rank test, two-tailed p = 0.219). (D) Spearman correlation of the pairwise distance to the amino acid subtype C consensus described in Fig 2 and RC scores over all variants tested (Non-parametric Spearman p = 0.0158, r = 0.4168.) The linear regression line is shown for visualization purposes.

Fig 6. Interferon-α resistance of TF and NT viruses.

(A) Spearman correlation of the replication measured by area under the curve (AUC) (y-axis) of each tested variant (black dots) and the replication (AUC) in the presence of interferon alpha (x-axis) (Non-parametric Spearman p < .0001, r = 0.8844). (B) Virus growth over 10 days in culture as measured by reverse transcriptase (RT) activity (DLU = digital light units) of the TF (blue), and NT variants (red) in the presence of IFN-α (dotted) and absence of IFN-α (solid lines) in an example pair 331. (C) RC scores in the presence of IFN-α were divided by the RC score in the absence of IFN-α for TF (blue) and selected NT (red) viruses with similar replication kinetics (Wilcoxon matched-pairs signed rank test, two tailed, p = 0.219). Subtype B TF (blue) and 6-month consensus (red) viruses are shown as controls on the right.