Evolution and recombination of genes encoding HIV-1 drug resistance and tropism during antiretroviral therapy

Abstract

Characterization of residual plasma virus during antiretroviral therapy (ART) is a high priority to improve understanding of HIV-1 pathogenesis and therapy. To understand the evolution of HIV-1 pol and env genes in viremic patients under selective pressure of ART, we performed longitudinal analyses of plasma-derived pol and env sequences from single HIV-1 genomes. We tested the hypotheses that drug resistance in pol was unrelated to changes in coreceptor usage (tropism), and that recombination played a role in evolution of viral strains. Recombinants were identified by using Bayesian and other computational methods. High-level genotypic resistance was seen in approximately 70% of X4 and R5 strains during ART. There was no significant association between resistance and tropism. Each patient displayed at least one recombinant encompassing env and representing a change in predicted tropism. These data suggest that, in addition to mutation, recombination can play a significant role in shaping HIV-1 evolution.

Figure 1. Phylogenetic trees of HIV-1 from patient WC4

(A) Protease and RT genes from plasma. The labels in red indicate HIV-1 variants predicted to be R5-tropic based on the V3 sequence; branches in blue indicate X4-tropic variants. The dates of the serial sequences are indicated for each variant in its identifier. Each branch represents a variant that is listed in Table 1A. (B) Phylogenetic tree of HIV-1 gp120 gene from plasma.

Figure 2. Phylogenetic trees of HIV-1 from patient WC2

(A) Protease and RT genes from plasma. The labels in red indicate HIV-1 variants predicted to be R5-tropic based on the V3 sequence; branches in blue indicate X4-tropic variants. The dates of the serial sequences are indicated for each variant in its identifier. Each branch represents a variant that is listed in Table 1B. (B) Phylogenetic tree of HIV-1 gp120 gene from plasma.

Figure 3. HIV-1 recombination in patient WC4

(A) Recombinant WC4P0397-8 and its most likely parental sequences are shown in the table at the top of the figure with predicted tropism and drug resistance mutations. (B) A Bayesian analysis of the envelope region of HIV-1 DNA sequences is shown below the table. Sequences that are predicted to be R5-tropic are shown in red; sequences predicted to be X4-tropic are in blue. An informative site analysis with similar color coding is shown at the bottom of the figure. (C) SimPlot of sequences of the envelope region of recombinant WC4P0397-8 and its most likely parentals. (D) Replication capacity assay of the recombinant WC4P0397-8 and its parentals in comparison to the control HIV-1 strain pNL4-3.

Figure 4. Replication capacity (RC) assay

The RC assay was based on transfection of a 10kb chimeric molecular clone, which was constructed by using ligation-mediated recombination PCR. HIV-1 pNL4-3 was used as a backbone with insertion of the entire pol gene amplified from plasma-derived virus by RT-PCR.

Figure 5. Additional HIV-1 recombination in patient WC4

(A) Recombinant WC4P0896-8 and its most likely parental sequences are shown in a table with predicted tropism and drug resistance mutations. (B) SimPlot of pol and env sequences of HIV-1 DNA fragments is shown below the table. (C) A sequence alignment of recombinant WC4P0896-8 and its most likely parental sequences in portions of pol and gp120 is shown below the SimPlot.

Figure 6. HIV-1 recombination in patient WC2

(A) Recombinant WC2P0896-4 and its most likely parental sequences are shown in the table with predicted tropism and drug resistance mutations. (B) A Bayesian analysis of pol and env sequences of HIV-1 DNA is shown below the table. Sequences that are predicted to be R5-tropic are shown in red; sequences predicted to be X4-tropic are in blue. An informative site analysis with similar color coding is shown at the bottom of the figure. Tau (τ) represents the marginal posterior probabilities of the most probable tree topology. That is, the probability of the putative recombinant clustering with tree i at each position. For this case, we used a 5 taxa tree so there is a topology (which is called “both parentals” which refers to the recombinant clustering with parentals 1 and 2 which had higher posterior support than (1,5) or (2,5). This is most likely due to the fact that both of the parental strains are very similar in this region and we could not distinguish between parentals 1 and 2. (C) SimPlot of pol and env sequences of the recombinant WC2P0896-4 and its parentals. The window size is 300bp, and the step size is 20bp. (D) Replication capacity assay of the recombinant WC2P0896-4 and its parentals in comparison to the control HIV-1 strain pNL4-3.

Figure 7. Analysis of limiting dilution PCR using a parametric model

(A) The sensitivity curve from the parametric model is shown in the solid line, and the empirical sensitivity of PCR at each dilution is shown in circles. (B) End-point limiting dilution PCR. Four HIV-1 strains that differ in restriction fragment length polymorphisms (RFLP) when their PCR product was digested by the restriction enzyme Nhe I (HIV-1 strains LAV, SF162, RF-V82F/I84V, and patient donor E) were mixed in known quantities. The DNA was subjected to 1% agarose gel electrophoresis. The left end of the gel (a: lanes1–5) shows the 4 HIV-1 strains after a minimal 1/50 dilution. The right end of the gel (b: lanes 6–14) illustrates an end-point dilution (1/500 dilution of the cDNA mixture), yielding a positive PCR in less than 40% of equivalent dilutions. (C) RFLP. The diluted cDNA was digested with Nhe I and subjected to 1% agarose gel electrophoresis. Lanes 1–5 (a) illustrate the gel pattern of lanes containing the mixed strains at the 1/50 dilution after digestion by Nhe I . Lanes 6–14(b) indicate, based on the gel pattern after Nhe I digestion, that the final 1/500 dilutions of the original mixture of 4 strains contained only one viral strain per lane.