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Background:  Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes has been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments neither shed light on the long-term in vivo dynamics of episomal cDNA, nor recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of a M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies.

Methods:  This study was conducted using archival patient samples from the AIDS Clinical Trials Group (ACTG) Protocol #306. Pereipheral blood mononuclear cells (PBMC) were obtained from treatment-naïve individuals at various intervals after initiating dual nucleoside analog therapy. Plasma HIV-1 RNA measurements were determined for most patients for weeks 2, 4, 8, 12, 16, 20, 24, 28, 36, 44, and 48 post-therapy initiation. We followed the emergence of drug-resistant mutations in episomal and proviral DNA in 11 patients who had received zidovudine (ZDV) plus lamivudine (3TC).  Under this regimen, resistance to 3TC develops rapidly based on a methionine-to-valine substitution at codon 184 (M184V) of reverse-transcriptase (RT). Polymerase chain reaction (PCR) primers targeting episomal and proviral sequences were used to specifically amplify RT from the respective templates. Amplification products from cellular DNA of archived PBMC that had been obtained at various intervals post-therapy initiation were sequenced directly to identify amino acid 184 of RT.

Results:  We demonstrate that during acquisition of drug resistance wild-type (WT) episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of WT with M184V-containing episomes occurred, while proviruses remained WT.

Conclusions:  Episomal viral cDNAs are labile, and hence are valid indicators of ongoing viral replication in vivo. The replacement of WT episomes by M184V-containing episomes while proviral sequences remained WT indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself.

Keywords: 2 LTR circle; viral reservoir; HAART