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Abstract
BackgroundAn essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300.ResultsIn this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process.ConclusionsThe results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.
Highlights
An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome
HIV-1 IN is acetylated by GCN5 To examine whether IN is acetylated by GCN5, in vitro acetylation assays were performed with recombinant IN and GCN5, both purified as GST fusion proteins
To define which region of IN is acetylated by GCN5, GST-IN fragments with progressive deletions starting from the C-terminus were used as substrates in in vitro acetylation assays, and the corresponding acetylation signals in the autoradiograms were evaluated by densitometric analysis (Figure 1B, left histogram)
Summary
An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300. Integration of reverse transcribed HIV-1 DNA into the cellular genome is catalyzed by the viral IN protein. HATs are enzymes able to transfer acetyl groups from acetyl coenzyme A (acetylCoA) to specific lysine residues within the N-terminal tails of nucleosomal histones, leading to chromatin decondensation and transcriptional activation [3,4]. HATs can acetylate non-histone substrates, such as transcription factors and other nuclear proteins, as well as cytoskeletal components, metabolic enzymes and signalling regulators in the cytoplasm [5].
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