Abstract
BackgroundGenomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation.ResultsHere, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication.ConclusionsOur data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0153-9) contains supplementary material, which is available to authorized users.
Highlights
Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir
Postintegration latency probably plays the dominant role in HIV-1 persistence, but pre-integration latency, which involves unintegrated viral DNA, may be relevant in vivo during quiescent CD4+ T cell infection, in which the virus persists as unintegrated viral DNA that is partially transcribed before cell activation [4,5,6]
After RAL removal, a decrease in the 2-LTR circles (2-LTRc) amount leading to a linear intermediate that is subsequently followed by new integration events
Summary
An obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. In infected cells, including resting CD4+ T cells, unintegrated viral genomes consist of the linear form Regarding their relative abundance, viral DNA forms can be ranked: unintegrated linear DNA (DNAL) > integrated provirus (DNAi) > 1-LTRc > 2-LTRc [7]. It is important to note that the repartition of viral genomes is dynamic during the course of infection and is dependent of viral conditions of infections such as mutations in the viral proteins or addition of compounds targeting viral or cellular proteins.
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