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.

Our former report showed that HIV-1 integration is positively regulated by the histone acetyltransferase (HAT) p300. In this study we demonstrate that another cellular HAT, GCN5, acetylates integrase leading to enhanced 3'-end processing and strand transfer activities. GCN5 plays a role during the integration step of the replication cycle as demonstrated by reduced infectivity due to lower provirus formation in cells silenced for GCN5. Within the Cterminus of integrase four lysines (K258, K264, K266 and K273) are targeted by GCN5 acetylation, three of which (K258, K264, K266) are also modified by p300. A viral replication analysis of HIV-1 viral clones carrying substitutions in lysines targeted by both GCN5 and p300 or exclusively by GCN5, demonstrated that these lysines are required for efficient viral integration. These results should clarify a recent debate raised on the role of these lysines during HIV-1 replication. In addition a comparative analysis of the replication efficiency of these viral clones demonstrate that, even though the lysines commonly targeted by both GCN5 and p300 are necessary for efficient integration, the lysine exclusively modified by GCN5 (K256) does not affect virus integration. In conclusion this study further demonstrates the relevance of integrase acetylation, which results from the catalytic activity of multiple HATs during the viral life cycle.

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a ‘protein delivery approach’ offers a new tool for developing novel strategies for genome modification and gene therapy applications.

Post-translational modifications, such as acetylation, dynamically modulate the chemical and structural properties of proteins generating new protein-protein interfaces. HIV-1 integrase is acetylated by p300 at three specific lysines located in the carboxy terminal domain. In the attempt to understand how acetylation modifies the integration event, we have searched for cellular cofactors that may specifically require acetylation to bind integrase.To this aim a tethered catalysis system has been exploited to perform a yeast two-hybrid screening. In this assay an integrase constitutively acetylated by fusion with the HAT catalytic domain of p300, was used as a ''bait'' to screen a human T-cell cDNA library. One of the identified binding factors was KAP1, which showed a higher affinity to integrase following its acetylation. This affinity was confirmed by either pull down assays and in vivo co-immunoprecitation in 293T cells. To evaluate the role of KAP1 during HIV-1 life cycle, infections were performed in HeLa and 293T cells transiently and stably silenced for KAP1. Interestingly, the infectivity was 3-10 fold higher than control cells and the analysis of the DNA forms showed a specific enhancement at the level of integration. In a reciprocal experiment overexpression of KAP1 showed a reduction of infectivity by a 50% decrease in integration.Since integrase activity is positively regulated by acetylation, we then performed experiments to explore whether KAP1 inhibition of viral integration might correlate with modulation of integrase acetylation levels. We demonstrated that KAP1 binding to acetylated integrase indeed induces integrase deacetylation through HDAC1 complex formation. Finally, HDAC1 complex formation is a requirement for KAP1 viral inhibition since no HIV-1 restriction can be observed in cell silenced for HDAC1.In conclusion, this study reports that KAP1, recently described to restrict M-MLV infectivity in embryonic stem cells at the level of viral transcription, inhibits HIV-1 through a novel mechanism targeting the integration step.from Frontiers of Retrovirology: Complex retroviruses, retroelements and their hosts