Background: Little is known about HIV-1 provirus expression in patients on suppressive ART. Characterizing cell-associated RNA (CAR) sequences in single cells can reveal their relationship to proviral populations and to persistent viremia. Here, we describe a new, sensitive method to assess the genetics of HIV CAR in viremic and virologically suppressed patients.
Methods: HIV-1 CAR was extracted from 4-6 separate aliquots of PBMCs from 3 patients using methods that were verified (by spiking known quantities of ACH2 cells into HIV negative PBMCs) to have high recovery of HIV-1 DNA and RNA. DNase-treatment and cDNA synthesis were optimized to ensure complete degradation of HIV DNA and high efficiency of long cDNA synthesis. Products were diluted to <1 HIV cDNA molecule/reaction, amplified, and sequenced (CAR-SGS). In patients who initiated ART with high HIV diversity, identical RNA sequences from the same aliquot of PBMCs were assumed to be derived from the same cell expressing HIV RNA, while identical sequences in different aliquots were assumed to be derived from clonally expanded, expressing cells. Single-genome sequences of proviral DNA and plasma HIV-1 RNA were compared to CAR sequences using standard bioinformatics methods.
Results: We developed and optimized methods for CAR-SGS to be used to profile cellular HIV-1 RNA expression in patients. 3 patients were studied: 1 was untreated and viremic and 2 were suppressed on ART. An average of 41 single cells per patient was analyzed. We found 53% of the HIV expressing cells in the viremic patient to be “high producers” (more than one RNA molecule detected) and 30% to be high producers in the suppressed patients. The diversity of the total RNA populations both before and during ART was ~1% in pro-pol, and G to A hypermutants were detected in the suppressed patients at similar levels to their proviral DNA populations (20% and 22%). We also detected HIV-1 expression from clonally expanded populations in one suppressed patient.
Conclusions: A new method to characterize the expression of HIV-1 proviruses that persist during ART reveals high levels of expression of RNA from infected individuals as well as clonally expanded cells. Further studies are needed to determine if HIV-1 expression results from spontaneous reactivation from latency or continuous low-level virus transcription and if these cells can be the source of viral rebound when ART is interrupted.