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The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr.

ABSTRACT: Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.

SUBMITTER: Dupont L

PROVIDER: S-EPMC8118623 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Silencing of unintegrated HIV-1 by SLF2 and the SMC5/6 complex

Project description:Silencing of nuclear DNA is an essential feature of the innate immune response to invading pathogens. Early in lentiviral infection, unintegrated viral cDNA accumulates in the nucleus, yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonises cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library, identifying SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. Using ATAC-seq, we show that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines an evolutionarily conserved antagonism by Vpr.

2021-03-01 | GSE156630 | GEO

Silencing of unintegrated HIV-1 by SLF2 and the SMC5/6 complex

Project description:Silencing of unintegrated HIV-1 by SLF2 and the SMC5/6 complex

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PTEN Mediates the Silencing of Unintegrated HIV-1 DNA.

Project description:The integration of viral DNA into a host genome is an important step in HIV-1 replication. However, due to the high failure rate of integration, the majority of viral DNA exists in an unintegrated state during HIV-1 infection. In contrast to the robust expression from integrated viral DNA, unintegrated HIV-1 DNA is very poorly transcribed in infected cells, but the molecular machinery responsible for the silencing of unintegrated HIV-1 DNA remains poorly characterized. In this study, we sought to characterize new host factors for the inhibition of expression from unintegrated HIV-1 DNA. A genome-wide CRISPR-Cas9 knockout screening revealed the essential role of phosphatase and tensin homolog (PTEN) in the silencing of unintegrated HIV-1 DNA. PTEN's phosphatase activity negatively regulates the PI3K-Akt pathway to inhibit the transcription from unintegrated HIV-1 DNA. The knockout (KO) of PTEN or inhibition of PTEN's phosphatase activity by point mutagenesis activates Akt by phosphorylation and enhances the transcription from unintegrated HIV-1 DNA. Inhibition of the PI3K-Akt pathway by Akt inhibitor in PTEN-KO cells restores the silencing of unintegrated HIV-1 DNA. Transcriptional factors (NF-κB, Sp1, and AP-1) are important for the activation of unintegrated HIV-1 DNA in PTEN-KO cells. Finally, the knockout of PTEN increases the levels of active epigenetic marks (H3ac and H3K4me3) and the recruitment of PolII on unintegrated HIV-1 DNA chromatin. Our experiments reveal that PTEN targets transcription factors (NF-κB, Sp1, and AP-1) by negatively regulating the PI3K-Akt pathway to promote the silencing of unintegrated HIV-1 DNA.

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Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

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Coordination of DNA damage responses via the Smc5/Smc6 complex.

Project description:The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.

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Project description:DNA polymerase epsilon (Pol ?) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ? catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability.

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Patients on HAART often have an excess of unintegrated HIV DNA: implications for monitoring reservoirs.

Project description:HIV establishes a latent reservoir early in infection that is resistant to anti-retroviral therapy and has a slow rate of decay. It is thought that the majority of HIV DNA in treated patients is integrated since unintegrated HIV DNA appears to be unstable. Thus, to monitor the HIV latent reservoir, total HIV DNA is commonly measured in PBMC from infected individuals. We investigated how often total approaches integrated HIV DNA in treated patients. To do this, we first assessed how accurate our integration assay is and determined the error in our measurements of total and integrated HIV DNA. We demonstrated an excess of total over integrated HIV DNA was present in a subset of patients, suggesting that measurements of total HIV DNA do not always correlate to the level of integration. Determining the cause of this excess and its frequency may have important implications for understanding HIV latent reservoir maintenance.

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Human cohesin compacts DNA by loop extrusion.

Project description:Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

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NP220 mediates silencing of unintegrated retroviral DNA.

Project description:The entry of foreign DNA into many mammalian cell types triggers the innate immune system, a complex set of responses to prevent infection by pathogens. One aspect of the response is the potent epigenetic silencing of incoming viral DNAs1, including the extrachromosomal DNAs that are formed immediately after infection by retroviruses. These unintegrated viral DNAs are very poorly transcribed in all cells, even in permissive cells, in contrast to the robust expression that is observed after viral integration2-5. The factors that are responsible for this low expression have not yet been identified. Here we performed a genome-wide CRISPR-Cas9 screen for genes that are required for silencing an integrase-deficient MLV-GFP reporter virus to explore the mechanisms responsible for repression of unintegrated viral DNAs in human cells. Our screen identified the DNA-binding protein NP220, the three proteins (MPP8, TASOR and PPHLN1) that comprise the HUSH complex-which silences proviruses in heterochromatin6 and retrotransposons7,8-the histone methyltransferase SETDB1, and other host factors that are required for silencing. Further tests by chromatin immunoprecipitation showed that NP220 is the key protein that recruits the HUSH complex, SETDB1 and the histone deacetylases HDAC1 and HDAC4 to silence the unintegrated retroviral DNA. Knockout of NP220 accelerates the replication of retroviruses. These experiments identify the molecular machinery that silences extrachromosomal retroviral DNA.

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Bacterial DNA Protects Monocytic Cells against HIV-Vpr-Induced Mitochondrial Membrane Depolarization.

Project description:Monocytes and macrophages are important HIV reservoirs, as they exhibit marked resistance to apoptosis upon infection. However, the mechanism underlying resistance to apoptosis in these cells is poorly understood. Using HIV-viral protein R-52-96 aa peptide (Vpr), we show that primary monocytes and THP-1 cells treated with Vpr are highly susceptible to mitochondrial depolarization, but develop resistance following stimulation with bacterial DNA or CpG oligodeoxynucleotide. We have shown that Vpr-induced mitochondrial depolarization is mediated by TNFR-associated factor-1 (TRAF-1) and TRAF-2 degradation and subsequent activation of caspase-8, Bid, and Bax. To provide the mechanism governing such resistance to mitochondrial depolarization, our results show that prior stimulation with CpG oligodeoxynucleotide or Escherichia coli DNA prevented: 1) TRAF-1/2 downregulation; 2) activation of caspase-8, Bid, and Bax; and 3) subsequent mitochondrial depolarization and release of apoptosis-inducing factor and cytochrome c Furthermore, this protection was mediated by upregulation of antiapoptotic protein (c-IAP-2) through calmodulin-dependent kinase-II activation. Thus, c-IAP-2 may prevent Vpr-mediated mitochondrial depolarization through stabilizing TRAF-1/2 expression and sequential inhibition of caspase-8, Bid, and Bax.

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