Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA

Abstract

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.

The HIV-1 Integrase C-Terminal domain induces TAR RNA structural changes promoting Tat binding

“…Noteworthy, the competition of Tat ARM with IN for TAR binding has also been reported elsewhere 8 . Our data are also fully consistent with recent evidences of the implication of IN in the transcription of the provirus at early times after integration and in a Tat-independent manner 32 . The authors found that mutation of four lysine residues within the IN-CTD dramatically reduced proviral transcription since all of them are involved in the binding of IN to the viral RNA 8,26 .…”

“…b Structural model of TAR RNA obtained with RNA Fold WebServer 63 64 predicting a minimum free energy of −29.60 kcal/mol. c Representative native 6% polyacrylamide gel illustrating the Electrophoretic mobility shift assay (EMSA) showing the interaction of IN-FLm with TAR RNA or an unstructured RNA 50-mer (AG (50) -mer) labelled with 32 P (black star). The RNA substrates (50 nM) were incubated with various concentrations of IN-FLm or without protein (TAR RNA: 0; 100, 200, 400 nM of IN-FLm; AG (50) -mer RNA: 0 or 400 nM of IN-FLm).…”

“…In order to verify that each RNA mutant assumed the expected secondary structure each model was analyzed with Fold WebServer 63,64 b EMSA assay illustrating the interaction of IN-CTD and IN-CTD-ΔCT with TAR wild type and mutants. The RNA substrates (50 nM) are labelled with 32 P (black star) and incubated with increasing concentrations of proteins (0; 100, 200, 400 and 800 nM) under the conditions described in ‘Materials and Methods’ section. c Graph showing the fractions of RNA bound by IN as a function of IN concentration.…”

“…Crosslinking-immuno-precipitation sequencing (CLIP-seq) experiments have identified the TAR RNA sequence as a major binding site of HIV-1 IN on the gRNA 8 . This observation, together with the recent finding of HIV-1 IN involvement in proviral transcription 32 , prompted us to study the interaction of IN with TAR RNA and its interplay with the Tat protein. Our results revealed that despite the apparent lack of structural specificity of IN in vitro , the CT flexible tail discriminates for the proper TAR apical stem-loop.…”

“…Recently, a new role has been proposed for HIV-1 IN during proviral transcription at early times after integration. In fact, after strand transfer, the IN remains bound to DNA and directly plays a role in proviral transcription, depending on its post-translational modifications of specific residues within the CTD 32 .…”