A9 A method to obtain full-length HIV proviral sequences and their sites of integration

Accurate definition of the HIV-1 reservoir on antiretroviral therapy (ART) is of paramount importance to the development of curative strategies. Much of this reservoir is derived from clonal expansion of latently infected CD4+ T cells. Methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis (ISA). However, current technologies do not link the intact proviruses detected by NFL-SGS to their sites of integration. Therefore, we developed a method to obtain both near full-length single-proviral sequences and their sites of integration. We call our method full-length integrated proviral single-genome sequencing (FLIP-SGS). Genomic DNA from ACH2 and CEM cells mixed at 1:1,000, or patient samples were diluted to a single proviral endpoint. An in-house, optimized whole genome amplification (WGA) method was performed on wells at the endpoint, generating multiple copies of all DNA molecules within each well. The number of proviral copies after WGA was determined by droplet digital PCR targeting the long terminal region (LTR). Forty per cent of each WGA reaction was used to obtain the provirus–host integration sites with ISA (linker ligation, nested PCR, and Illumina sequencing). The remaining fraction was used to amplify the full-length proviruses in four overlapping fragments (LTR-pol, gag-int, int-env, and env-LTR) for Sanger sequencing. WGA performed on the endpoint-diluted ACH2:CEM DNA amplified single-copy HIV-1 proviral templates greater than 500-fold, making it possible to obtain unique integration sites from single proviruses in ACH2 cells, including one that was previously reported (in the NT5C3A gene on chromosome 7) and two that were not previously reported (in the EIF4ENIF1 gene of chromosome 22 and an unknown region of chromosome 6). Near full-length PCR amplification and Sanger sequencing was performed on proviruses integrated in the NT5C3A gene. FLIP-SGS was applied to peripheral blood mononuclear cells from one HIV-1 infected donor with viremia suppressed on ART and yielded integration sites of four genomes that appear to contain large internal deletions. We report a method for near full-length HIV-1 single-genome sequencing combined with host integration site detection that we call FLIP-SGS. This assay will further define clonal expansion of infected CD4+ T cells as a mechanism that maintains the HIV-1 reservoir and as the source of identical sequences observed during therapy and rebound, rather than from ongoing replication.