The role of integration and clonal expansion in HIV infection: live long and prosper

Abstract

Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.

Keywords: Clonal expansion; HIV persistence; HIV reservoirs; Proviral integration.

Fig. 1

Structural domains and function of HIV integrase

Fig. 2

Depiction of the integration of HIV proviral DNA into the host genome

Fig. 3

Linker mediated HIV integration site assay (ISA) workflow. Total genomic DNA is first extracted then randomly sheared by Covaris sonification into 300–500 bp fragments. Sheared fragments are end repaired and a single dA overhang is added, then linkers containing a single T overhang are ligated onto the sheared ends (red). The pop out displays the PCR amplification strategy to selectively amplify integration sites. Primers that are complementary to the 5′ HIV LTR in U3 (dark grey arrow) and the 3′ HIV LTR in U5 (light grey arrow) are combined with linker specific primers (red arrows). The resulting amplicons contain linker sequence, the random breakpoint (BP), and the HIV/host junction sequence at the integration site (IS). The amplicons are then subjected to Illumina Miseq paired end sequencing. Sequences obtained are run through a stringent bioinformatics pipeline to map the location of the integrated provirus against a reference host genome and to determine the distance to breakpoint. Identical integration sites from amplicons with different break points in the host genome are the result of clonally expanded cells, whereas identical integration sites from amplicons with identical break point distances arose during PCR amplification

Fig. 4

HIV integration site loop amplification (ISLA) assay workflow. HIV DNA copy numbers are quantified from extracted nucleic acid and diluted to an endpoint prior to linear extension using primers in HIV env and HIV nef, then random decamers (blue) tailed with an HIV LTR U5-specific sequence (red) are annealed to the linear template and extended, the single stranded DNA downstream of the random decamer primer is removed and the U5-specific region anneals to its complementary sequence in the HIV LTR forming a loop which is then amplified, the resulting loop contains U5 sequence that is flanked by the host genome, using primers complementary to U5 the integration site can be mapped. Integration sites identified more than once indicate clonal expansion