(736a) Single Proviral Sequencing of Latent HIV-1 Proviruses Using Droplet Microfluidics | AIChE

(736a) Single Proviral Sequencing of Latent HIV-1 Proviruses Using Droplet Microfluidics


Abate, A., University of California San Francisco
Reservoirs in HIV infection that lead to persistent viremia in patients on antiretroviral therapy (ART) are the main barriers to a cure. The major HIV reservoir is latent proviruses persist in resting memory CD4+ T cells. Proviral sequence and its integration site in resting CD4+ T cells may provide important clues in mechanisms contributing to HIV persistence. HIV provirus are highly heterogeneous, full-length single genome sequencing would be optimal to detect low frequency variants. The low viral load in patients on ART (approximately one cell in every thousand among peripheral blood CD4 T cells, as measured by quantitative PCR of virus DNA) and the small genome size (<10 kb, compared to 3.2 Gb of human genome) of HIV bring the needs to enrich the provirus genome for HIV single provirus sequencing. However, due to the extreme rarity, complexity and lack of surface marker of latently HIV-infected CD4 T cells, the identification and manipulation of these cells in blood or lymphoid tissue samples from HIV-infected patients has previously been infeasible.

Here, we develop a droplet microfluidic based single provirus sequencing (SiP-seq) method to sequence HIV genome together with it’s adjacent human genome from latently infected T cells. Genomic DNA fragments are compartmentalized and amplified in isolated pico-liter water-in-oil emulsions, followed by PCR-activated sorting of them based on the presence of HIV sequence. The amplification of genome in individual pico-liter droplets also enables maximum amplification uniformity and minimal contamination. We have validated the method using J-Lat cell line (a Jurkat based cell line, with each cell containing a full-length integrated HIV-1 genome with a frameshift in Envand GFP in place of the Nef). We have then sorted and sequenced single HIV genomes as well as its integration sites in latently HIV-infected CD4 T cells from ART-suppressed patients. We showed the first true full-length sequencing of HIV provirus as well as aberrant HIV-1 integrations from ART-suppressed patients. Our approach allows isolation and sequencing large number of inserted single provirus from latently infected T cells, which will provide novel insights to the genetic mechanisms regulating HIV persistence.