Background
Residual viremia (RV) persists despite years of effective ART due to the daily activation of latently infected cells. Some people on long-term ART develop nonsuppressible viremia (NSV), characterized by persistently detectable HIV RNA that complicates clinical care, raising concerns for virological failure and HIV transmission. Recent work from our group and others showed that proviruses with defects in the 5’Leader (5’L) can also cause NSV. However, their overall contribution to persistent viremia remains unknown.
Methods
We enrolled 30 participants with intermittent or persistent plasma HIV RNA above the limit of quantification by clinical assays (20-1500 copies/mL) despite no history of adherence issues. We isolated virus-associated RNA by plasma centrifugation and performed single genome sequencing covering the U5-gag and protease-reverse transcriptase (P6-RT) regions. Sequences were analyzed for drug-resistance mutations, clonality, and 5’L defects. To benchmark the frequency of major splicing donor (MSD) mutations pre-ART, we used 2238 5’L RNA sequences from the LANL HIV database. The impact of 5’L defects on viral infectivity was tested by site-directed mutagenesis of pNL4-3.
Results
We obtained 376 U5-gag and 358 P6-RT HIV RNA sequences from 29 participants with a median of 28 sequences per participant. No drug-resistance mutations relevant for current ART were detected. On average, 90% of sequences were identical, with a median of 2 unique variants per participant. Estimates of clonality by U5-gag and P6-RT strongly correlated (Spearman r=0.77, P<0.0001). We detected HIV RNA with 5’L defects in 27/29 participants, contributing to an average of 78% (sd±34%) of defective sequences per participant. Among 65 unique 5’L variants recovered, 22 were intact, 18 had deletions of 2-98 nucleotides involving the MSD, and 25 had single mutations in three conserved positions (HXB2 G744, T745, G748) necessary for MSD-spliceosome binding. These mutations abolished viral replication in vitro and were significantly enriched in participants with NSV compared to the pre-ART sequences (Chi-square, p<0.00001). Conversely, the polymorphic T742C substitution showed no significant difference in frequency (p=0.35).
Conclusions
HIV RNA contributing to NSV is highly clonal and enriched in narrowly focused 5’L defects that prevent de novo invention events. These findings can inform the clinical management of NSV and support the development of assays to distinguish intact from defective HIV RNA in plasma.
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