Circular Viral DNA and Anomalous Junction Sequence in PBMC of HIV-Infected Individuals with No Detectable Plasma HIV RNA

Abstract

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels <50 copies/ml of blood had detectable 2-LTR DNA. Sequencing of the products identified normal LTR--LTR junctions in the minority of cases with the majority containing anomalies including deletions and insertions. The persistence of HIV ccDNA in patients with no detectable plasma RNA could be consistent with ongoing de novo infection of dividing cells or with stability of this form of DNA in nondividing cells.

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys

“…Indeed, in the vaccinated and protected monkeys from this study, viral load (either cell-or plasma-associated) was persistently low or undetectable and CD4 T-cell decline did not occur. These data were confirmed by the determination of unintegrated forms of DNA, a very sensitive marker for active viral replication [59] as compared to plasma viremia [44,45]. In our study, detection of circular E-DNA correlated with the presence of a detectable plasma viremia during the acute phase of infection, in agreement with previous reports in both the HIV [60,61] and the SIV model [62].…”

“…For analysis of viral extrachromosomal circular DNA (E-DNA), DNA was extracted from lymphocytes ((2-5) × 10 6 ) using the QIAprep Spin Miniprep Kit in order to enrich for the low molecular weight DNA fraction, and analyzed by PCR for the presence of circular forms of viral DNA as described [44,45]. Twenty microliters of the total volume (50 l) were denaturated at 95 • C for 5 min, and subjected to 40 cycles of PCR with an annealing temperature of 60 • C for 30 s, an extension temperature of 72 • C for 30 s and a denaturation temperature of 95 • C for 30 s, using primers FORN15 -GTGACTCCACGCTTGTTTGC-3 (forward), located in the R LTR region, and REVN15 -CTCCTGTGC-CTCATCTGATACA-3 (reverse), mapping in the U3 LTR region of the SIVmac239.…”

“…To better evaluate virus replication, E-DNA, a parameter more sensitive than the evaluation of plasma viremia [44,45], was measured in PBMCs of all macaques (with the exception of monkey 2) and compared with plasma viremia at early and/or late time points post-challenge (Fig. 1).…”

Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

“…Nevertheless, such a strategy requires further investigations to establish that HIV-1 circles can direct the formation of proteins in vivo and even infectious viral particles. If this is confirmed, it would indicate that cells containing viral DNA circles would constitute an additional and stable reservoir for HIV-1, as they are found in vivo (9,15,19,23,28,35) and as circles bearing two LTR were shown to persist in peripheral blood mononuclear cells from HIV-1-infected patients, even under the best of our currently available antiretroviral treatments (3).…”

An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

“…In vivo, this inefficiency of integration results in an accumulation of unintegrated HIV-1 DNA (uDNA) in blood CD4 ϩ T cells and monocytes (7,8,11,12), as well as in lymphoid tissue (8-10), brain (13,14), and untreated patients with high or low viral loads (15,16). During untreated chronic infection, the majority of infected cells have been reported to contain only unintegrated HIV-1 DNA (7).…”