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. 2014 Jul 11;345(6193):179-83.
doi: 10.1126/science.1254194. Epub 2014 Jun 26.

HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells

F Maldarelli  1 , X Wu  2 , L Su  2 , F R Simonetti  3 , W Shao  2 , S Hill  1 , J Spindler  1 , A L Ferris  1 , J W Mellors  4 , M F Kearney  1 , J M Coffin  5 , S H Hughes  6
Affiliations

HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells

F Maldarelli et al. Science. .

Abstract

The persistence of HIV-infected cells in individuals on suppressive combination antiretroviral therapy (cART) presents a major barrier for curing HIV infections. HIV integrates its DNA into many sites in the host genome; we identified 2410 integration sites in peripheral blood lymphocytes of five infected individuals on cART. About 40% of the integrations were in clonally expanded cells. Approximately 50% of the infected cells in one patient were from a single clone, and some clones persisted for many years. There were multiple independent integrations in several genes, including MKL2 and BACH2; many of these integrations were in clonally expanded cells. Our findings show that HIV integration sites can play a critical role in expansion and persistence of HIV-infected cells.

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Figures

Fig. 1
Fig. 1. Long term cART reveals, the presence, in patients, at both the RNA and the DNA level, of HIV genomes that have identical sequences
(A to E) Single-genome sequences were obtained from plasma virion RNA or from PBMC DNA prior to or shortly after the initiation of cART, and after long-term cART. Sequences were aligned using Clustal W, and neighbor-joining trees were rooted on the consensus sequence of subgroup B HIV (). Virus populations from pretherapy samples, or samples taken shortly after the initiation of cART, are shown as open circles (red, virion RNA; black, PBMC DNA); populations after prolonged cART, as closed circles (red, virion RNA; black, PBMC DNA). In patient 1, the black dot, marked by the arrow, represents DNA sequences from the provirus in a clone of expanded cells, whose integration site could not be mapped (“ambiguous”, Fig. 2). Short branches and low bootstrap values on major nodes of the trees support a lack of divergence between preor early therapy sequences and populations of identical sequences after prolonged cART (). To avoid distorting the trees, all hypermutant sequences were removed for the analysis shown in the figure. * Denotes the trees from which G>A hypermutant sequences were removed.
Fig. 2
Fig. 2. Distribution of integration sites in the five patients
A total of 2410 integration sites were obtained from PBMCs or negatively selected CD4+ cells from the five patients. Genes in which we isolated a particular integration site 7 or more times are shown. The table shows which patient harbored each of these expanded clones, how many times the different integrations sites were isolated, and the fraction of the infected cells the expanded clone represents in the patient. The MKL2 clones marked a, b, c, and d correspond to the integration sites marked a, b, c, and d in Fig. 3. One provirus in a highly expanded clone in patient 1 was in a sequence that could not be unambiguously assigned in the human genome (denoted “ambiguous”). Our standard analysis (presented here) underestimated the fraction of the infected cells in patient 3 that had an integration site in HORMAD2; see text and ().
Fig. 3
Fig. 3. Integration sites in the MKL2 and BACH2 genes in patient 1 after 11.4 years of cART
(A) There were 15 distinct integration sites (blue arrows) in a small region of the MKL2 gene in patient 1. The arrows denote the transcriptional orientation of each provirus. The circled arrows indicate integration sites in clonally expanded cells. The arrows marked a, b, c, and d correspond to the clones marked a, b, c, and d in Fig. 2. (B) There were 15 distinct integration sites in a small region of the BACH2 gene in patient 1 (blue arrows). Some of these integration sites were in clonally expanded cells (circled arrows). Panels A and B also show the HIV integration sites identified in the same two genes in acutely infected HeLa cells (total sites=248,658, brown arrows) and CD34+ cells (total sites=159,484, green arrows).
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