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The HUSH complex (composed of TASOR, MPP8 and periphilin) represses HIV-1 expression from its promoter by inducing both propagation of repressive epigenetic marks and degradation of the nascent transcript. Vpx from HIV-2, and Vpr proteins from some simian lentiviruses (SIVs), antagonize HUSH, thereby increasing proviral expression. The chromatin-remodelling MORC2 protein plays a critical role in the epigenetic silencing of host genes by HUSH. Here, we deciphered the role of MORC2 in retroviral silencing. We show that MORC2, in contrast to HUSH components, presents strong signatures of positive selection during primate evolution. Like HUSH, MORC2 represses proviral expression in two models of HIV-1 latency. However, while HUSH is degraded upon HIV-2 infection in a Vpx-dependent manner, MORC2 levels are increased, raising the question of a feedback control mechanism without HUSH. Upon infection with an HIV-1-derived virus, MORC2 and TASOR antiviral effects are interdependent. However, once the lentiviral DNA is integrated into the host genome, MORC2 may maintain the repression independently of HUSH. At the post-transcriptional level, both MORC2 and HUSH act in association with CNOT1 of the CCR4-NOT deadenylase complex and the TRAMP-like PAXT complex. Finally, MORC2, but not HUSH components, is expressed in primary quiescent CD4+ T cells. Altogether, our data highlight MORC2 as an HIV restriction factor and a chromatin remodelling protein operating both at the transcriptional and post-transcriptional levels. We speculate that MORC2 could serve as an immune gatekeeper following HUSH inactivation by Vpx and contribute to the maintenance of retroviral silencing in reservoir CD4+ T cells.Significance statementOne hurdle to HIV eradication is viral latency, which refers to the persistence of the virus in reservoir cells despite antiretroviral treatment. The HUSH complex represses HIV expression, once the viral genome is integrated into the host genome. HUSH activity on host genes depends on MORC2, a protein incriminated in the Charcot-Marie-Tooth neuronal disease. Here, we first show that MORC2 presents signs of evolutionary arms-races in primates. Furthermore, MORC2 contributes to HIV silencing in cooperation with HUSH, but also, likely without HUSH. Despite identified as a chromatin remodeler, MORC2 also works at a post-transcriptional level. Altogether, MORC2 appears as a host defense factor, which plays a role in HIV latency.

Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells., Author summary Human Immunodeficiency Virus (HIV) is still a major public health issue. The understanding of the molecular battle occurring during viral infection, between HIV components and cellular antiviral factors, the so-called restriction factors, is a key determinant for new treatment development. Namely, HIV auxiliary proteins are powerful to induce the downregulation of cellular restriction factors by hijacking the Ubiquitin/proteasome pathway, in order to facilitate the completion of a well-processed HIV replication cycle. For instance, HIV-2 Vpx eases reverse transcription in myeloid cells by counteracting the SAMDH1 restriction factor. More recently, we discovered the ability of Vpx to induce the degradation of the HUSH epigenetic repressor complex to favor in turn, the expression of the provirus. In this study, we uncovered the mechanisms by which Vpx antagonizes TASOR, the core subunit of the HUSH complex. We highlighted key differences between Vpx-induced TASOR and SAMHD1 degradation. These findings will help to propose strategies to study or to target either HUSH or SAMHD1, especially in myeloid cells where SAMHD1 restriction operates.

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, LINE-1, Retrotransposons and Retroelements or the integrated HIV provirus1–5. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of silencing mediated by HUSH is still poorly understood. Here, we show that TASOR depletion increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. By counteracting HUSH, Vpx from HIV-2 mimics TASOR depletion. With the use of a Yeast-Two-Hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex presents structural homology with HUSH. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Similarly, we show that TASOR interacts and cooperates with MTR4 and the exosome, in addition to CNOT1. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Furthermore, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.