New Assay Reveals Vast Excess of Defective over Intact HIV-1 Transcripts in Antiretroviral Therapy-Suppressed Individuals

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi RRE ), 3' defective (Psi RRE ), and 5' defective (Psi RRE ) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4 T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/10 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/10 cells; 5.6%) or intact (0.6 copies/10 cells;