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Postintegration transcriptional silencing of HIV-1 leads to the establishment of a pool of latently infected cells. In these cells, mechanisms controlling RNA Polymerase II (RNAPII) pausing and premature transcription termination (PTT) remain to be explored. Here, we found that the cleavage and polyadenylation (CPA) factor PCF11 represses HIV-1 expression independently of the other subunits of the CPA complex or the polyadenylation signal located at the 5' LTR. We show that PCF11 interacts with the RNAPII-binding protein WDR82. Knock-down of PCF11 or WDR82 reactivated HIV-1 expression in latently infected cells. To silence HIV-1 transcription, PCF11 and WDR82 are specifically recruited at the promoter-proximal region of the provirus in an interdependent manner. Codepletion of PCF11 and WDR82 indicated that they act on the same pathway to repress HIV expression. These findings reveal PCF11/WDR82 as a PTT complex silencing HIV-1 expression in latently infected cells. [ABSTRACT FROM AUTHOR]

Background: The recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m6A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m6A writers and readers on the biogenesis of HIV-1 RNAs. Results: We showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic writer down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear writer YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity. Conclusions: Our findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m6A nuclear reader YTHDC1 is required for efficient production of infectious viral particles. [ABSTRACT FROM AUTHOR]

Abstract Background HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood. Results Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization. Conclusions Our findings indicate that, shortly after viral entry, a significant portion of DNA–free IN that is distinct from active pre-integration complexes accumulates in the nucleus.

Abstract Background Integration of human immunodeficiency virus type 1 (HIV-1) into a host cell chromosome is an essential step under the control of the viral integrase (IN). Although this enzyme is necessary and sufficient to catalyze the integration reaction in vitro, cellular cofactors are involved in the process in vivo. The chromatin-associated factor LEDGF/p75 interacts with IN and promotes integration to transcription units of the host genome. HIV-1 IN also binds the karyopherin TNPO3, however the significance of this interaction during viral replication remains to be explored. Results Here we present a functional analysis of IN mutants impaired for LEDGF/p75 and TNPO3 interaction. Among them, IN W131A and IN Q168L, that were previously identified to be deficient for LEDGF/p75 interaction, were also partially impaired for TNPO3 binding. We observed that mutations abolishing IN ability to form tetramers resulted in a severe reduction in LEDGF/p75 binding. In sharp contrast, no correlation could be found between the ability of IN to multimerize and TNPO3 interaction. Most of the mutant viruses were essentially impaired for the integration step whereas the amount of 2-LTR circles, reflecting the nuclear import of the viral DNA, was not significantly affected. Conclusion Our functional analysis of HIV-1 IN mutants reveals distinct structural basis for TNPO3 interaction and suggests that the interaction between IN and TNPO3 is not a major determinant of nuclear import but could take place at a nuclear step prior to integration.

Summary The persistence of a latent reservoir containing transcriptionally silent, but replication-competent, integrated provirus is a serious challenge to HIV eradication. HIV integration is under the control of LEDGF/p75, the cellular cofactor of viral integrase. Investigating possible postintegration roles for LEDGF/p75, we find that LEDGF/p75 represses HIV expression in latently infected cells. LEDGF/p75 associated with two proteins involved in the control of gene expression and chromatin structure, Spt6 and Iws1, to form a stable complex. Iws1 plays a role in the establishment of latent infection, whereas Spt6 functions to recruit Iws1 and LEDGF/p75 to the silenced provirus and maintains histone occupancy at the HIV promoter. In latently infected cells, depletion of the complex results in reactivation of HIV expression Altogether, our results indicate that a complex containing LEDGF/p75, Iws1, and Spt6 participates in regulating postintegration steps of HIV latency. [ABSTRACT FROM AUTHOR]

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNFS/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNFS/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNFS/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNFS/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Inil as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNFS/Ini1 interferes with early steps of HIV-1 infection. [ABSTRACT FROM AUTHOR]

β-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that β-TrCP targets the oncogenic protein β-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from β-catenin containing the phosphorylated motif DpSGXXpS (P-β-Cat17-48) and β-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the β-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter β-catenin fragment with the β-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated β-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface. [ABSTRACT FROM AUTHOR]