Background
Most methods of amplifying and sequencing proviruses do not capture the flanking long terminal repeats (LTRs) because of technical difficulties related to sequence identity between these elements. Consequently, defective HIV-1 proviruses consisting of a single LTR (solo-LTR), a common proviral structure across retroviruses, are not detected and thus their frequency and significance are poorly understood. Here we developed a new method able to detect solo LTRs and characterize their abundance in PBMC samples from PWH on ART.
Methods
Several established methods and a new approach were used to sequence full-length proviruses in the largest HIV containing T cell clones in 3 adult PWH with detectable viremia on long-term ART (9-23 years). The methods included integration site analysis (ISA), multiple displacement amplification single genome sequencing (MDA-SGS), integration site qPCR (IS-qPCR), and a new droplet-based PCR method in which HIV-host “junction” primers are used to amplify full-length proviruses by nested PCR.
Results
Full-length amplification and sequencing of proviruses in 24 of the largest infected T cell clones in three PWH revealed 2 intact and 22 defective proviruses, of which 7 contained large internal deletions and 12 were solo-LTRs. MDA-SGS and ISA identified a provirus with internal HIV sequence, which included Psi, that had the same integration site as a clone carrying solo-LTR proviruses. This finding suggests that the progenitors of proviruses that undergo recombination to become solo LTRs may persist at low levels on ART.
Conclusions
Our results reveal that solo-LTRs are the most common proviral structure found in large HIV-infected cell clones in three PWH on ART. Using droplet-based PCR with HIV-host junction primers, several solo-LTRs were characterized that would have otherwise been undetectable. One provirus with internal sequence was found to share an integration site with a large clone carrying a solo-LTR, suggesting that the solo-LTR clone originated from an ancestral cell or cells carrying the provirus with internal sequence that underwent homologous recombination to generate a solo-LTR in a daughter clone. Identifying and exploiting the mechanisms that promote the formation of solo- LTRs could enable inactivation of proviruses with internal sequences, including intact proviruses.
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