Analysis of ex vivo HIV-1 infection in a controller-discordant couple

Abstract

Objectives: Elite controllers (EC) are a rare group of individuals living with HIV-1 who naturally control HIV-1 replication to levels below the limit of detection without antiretroviral therapy (ART) and rarely progress to AIDS. The mechanisms contributing to this control remain incompletely elucidated. In the present study, we have assessed whether cellular host factors could modulate HIV-1 replication post-entry in a controller-discordant couple living with HIV-1.

Methods: CD4 T cells from a controller-discordant couple, one partner being an EC and the other an HIV-1 progressor (PR), and healthy controls (HC) were isolated, activated and infected with VSV-G pseudotyped yellow fluorescent protein-encoding single-round HIV-1 virus (HIV-YFP). Viral reverse transcripts, 2-LTR circles and integrated proviral HIV-1 DNA were monitored by quantitative PCR (qPCR) and integration sites were analysed. We further measured LEDGF/p75 and p21 mRNA expression levels by qPCR.

Results: Infection of activated CD4 T cells with HIV-YFP was reduced in EC compared with the PR partner, and HC. Evaluation of viral DNA forms suggested a block after entry and during the early steps of HIV-1 reverse transcription in EC. The integration site distribution pattern in EC, PR and HC was similar. The p21 expression in CD4 T cells of EC was elevated compared with the PR or HC, in line with previous work.

Conclusions: Our study suggests a reduced permissiveness to HIV-1 infection of CD4 T cells from EC due to a block of HIV-1 replication after entry and before integration that might contribute to the EC phenotype in our patient.

Keywords: HIV, elite controllers, HIV eradication, integration.

Figure 1.

Analysis of ex vivo infection of a controller-discordant couple. (a) Percentage of YFP-positive CD4 T cells from EC and PR at day 2 and 7 after transduction with a serial dilution (1/10 and 1/20) of HIV-YFP; a representative experiment for multiple independent experiments (n=4), is shown. (b,c) LEDGF/p75 and p21 mRNA expression relative to beta-actin expression (mean, standard deviation) in stimulated CD4 T cells demonstrating only a mild increase in p21 in EC. Statistically significant differences using a one-way ANOVA are indicated with * (**: P=0.03; ***: P=0.0025). (d) Kinetics of viral intermediates (late reverse transcripts or Late RT, 2-LTR circles, integrated copies expressed relative to RNaseP) in CD4 T cells from EC, PR, or HC without efavirenz (EFV at 50xIC₅₀) or raltegravir (RAL at 50xIC₅₀) after transduction with HIV-YFP using qPCR demonstrating an early reduction in late RT products in EC compared with PR or HC. (e) For integration site analysis, cells were split and maintained for 7 days before determining the number of integrated copies and integration site analysis. Heat maps were developed to summarise relationships of proviral integration sites with genomic features using the receiver operating characteristic (ROC) area method [19). The analysed genomic features are mentioned on the left of the corresponding row of the heat map. Tile colour indicates whether a chosen feature is favoured (red, enrichment compared with random) or disfavoured (blue, depletion compared with random) for integration for the respective data sets relative to their MRCs, as detailed in the coloured ROC area scale at the bottom of the panel. The different data sets used are indicated above the columns. The *denote significant differences of HIV integration compared to the LEDGF/p75 knockdown cell line for the respective features (*: P<0.05; ***: P<0.001, using Wald statistics referred to a chi-squared distribution), dashes overlay control tiles. The naming of the genomic features is described in Brady et al. . TSS: transcription start site; EC: elite controller; HC: healthy controller; PR: progressor; YFP: yellow florescence protein.