Proviral sequencing suggests the majority of the HIV reservoir is expressed over time but significant decay is obscured by clonal expansion

After initiating antiretroviral therapy (ART), a rapid decline in plasma viremia is followed by reservoir stabilization. Viral outgrowth assay suggests the reservoir continues to decline slowly, but variation over time and among individuals complicates our understanding of selective pressures during ART. We used full-length sequencing to study more than 800 HIV proviruses of two subjects on ART at four time points over nine years to investigate the selection pressures influencing the dynamics of the reservoir. We found that intact as well as defective proviruses capable of significant protein expression decrease over time. Moreover, proviruses lacking genetic elements to promote viral protein expression, yet containing strong splice donor sequences increase relative to other defectives over time, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by clones generated by donor splice site-enhanced clonal expansion.