Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir

Abstract

The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.

Fig 1. Schematic of experimental approach.

(a) To perform quantitative viral outgrowth assays (VOA), donor CD4⁺ T cells (resting or total) were serially diluted, stimulated with PHA, and co-cultured with irradiated feeder cells and CD8-depleted allogeneic blasts from HIV-negative donors for 14 to 21 days. Single-genome sequencing (p6-PR-RT or near full-length) was performed on supernatants from p24-positive wells, and the sequences from the different wells were analyzed for identical matches. (b) Single-genome sequencing was performed on uncultured blood mononuclear cells to obtain p6-PR-RT sequences from HIV DNA and/or unspliced HIV RNA to search for identical sequence matches to RNA sequences from p24-positive VOA wells. (c) Schematic showing hypothetical examples of identical sequence matches between single-genome sequences from p24-positive wells, HIV DNA, and cell-associated HIV RNA analyzed by neighbor-joining distance analysis.

Fig 2. Neighbor-joining distance tree of sequences in p24-positive viral outgrowth assay wells and in HIV DNA sequences from blood mononuclear cells (Donor 1).

The tree was constructed using the neighbor-joining p-distance method and rooted to a subtype B consensus sequence. p6-PR-RT single-genome sequences were obtained from HIV DNA in total CD4⁺ T-cells. Hypermutant HIV DNA sequences are shown in dashed boxes. p6-PR-RT single-genome sequences were also obtained from independent, p24-positive viral outgrowth assay (VOA) wells performed using either total CD4⁺ T-cells or resting CD4⁺ T-cells. Different colored diamond symbols represent sequences from different p24-positive VOA wells. Identical p6-PR-RT sequences were recovered from six p24-positive VOA wells (red arrow), with confirmed matches of viral RNA by overlapping half-genome sequencing (*). These matching near full-length sequences appeared intact without large deletions, frame-shift mutations, or disabling stop codons. Note that the large groups of identical sequences from VOA wells are accompanied by multiple sequence variants that differ from the predominant sequence by 1–2 nucleotides, a result we attribute to mutations that arose during ex vivo virus replication and/or to errors introduced during in vitro cDNA synthesis.