Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex

Abstract

Drugs that inhibit HIV-1 replication and prevent progression to AIDS do not1 eliminate HIV-1 proviruses from the chromosomes of long-lived CD4 + memory T 2 cells. To escape eradication by these antiviral drugs, or by the host immune 3 system, HIV-1 exploits poorly defined host factors that silence provirus 4 transcription. These same factors, though, must be overcome by all retroviruses, 5 including HIV-1 and other primate immunodeficiency viruses, in order to activate 6 provirus transcription and produce new virus. Here we show that Vpx and Vpr, 7 proteins from a wide range of primate immunodeficiency viruses, activate 8 provirus transcription in human CD4 + T cells. Provirus activation required the 9 DCAF1 adaptor that links Vpx and Vpr to the CUL4A/B ubiquitin ligase complex, 10 but did not require degradation of SAMHD1, a well-characterized target of Vpx 11 and Vpr. A loss-of-function screen for transcription silencing factors that mimic 12 the effect of Vpx on provirus silencing identified all components of the Human 13 Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), 14 PPHLN1 (PERIPHILIN), and MORC2. Vpx associated with the HUSH complex 15 components and decreased steady-state levels of these proteins in a DCAF-16 dependent manner. Finally, vpx and FAM208A knockdown accelerated HIV-1 and 17 SIV MAC replication kinetics in CD4 + T cells to a similar extent, and HIV-2 replication 18 required either vpx or FAM208A disruption. These results demonstrate that the 19 HUSH complex restricts transcription of primate immunodeficiency viruses and 20 thereby contributes to provirus latency. To counteract this restriction and 21 activate provirus expression, primate immunodeficiency viruses encode Vpx and 22 Vpr proteins that degrade HUSH complex components. 23 2017), whose demonstration that cells may carry Rous sarcoma virus genetic 479 information in the absence of any infectious virus production provided support to the 480 proviral hypothesis. We thank Lisa Cavacini for anti-CD3 and anti-CD28 antibodies, and 481 Akio Adachi and Mikako Fujita for pGL-St and pGL-An. The following reagents were 482 obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH: J-Lat Tat-483

NP220 mediates silencing of unintegrated retroviral DNA

“…These findings define new functions for NP220 and the HUSH complex. The restriction is sufficiently strong that many viruses have evolved means to evade or inactivate this machinery: the Vpr and Vpx proteins from primate immunodeficiency viruses mediate degradation of the HUSH complex to relieve silencing of proviruses 15,16 , and the ICP0 gene of HSV-1 relieves HDAC-mediated silencing of viral DNA 17,18 . The silencing mechanisms and machinery uncovered here may have wide-ranging impacts on the design of non-integrating retroviral vectors for gene therapy and the gene expression following plasmid DNA transfection.…”

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing

“…The colocalization of these two protein complexes to promoters in differentiated cells may therefore be a remnant of the developmental program (Figure 5A) [1,52]. (2) We and others have demonstrated that the degree of transcriptional silencing by HUSH is promoter-specific [3,8,14,21], and extend this observation to PRC1.6. Intrinsic properties of select exogenous and endogenous promoters such as viral origin, paused RNA Polymerase II occupancy, or transcription factor binding likely contribute to localization or activity of PRC1.6 and the HUSH complex.…”

“…HUSH silencing of lentivectors is modulated at least in part by the promoter that drives expression of the transgene [3,8,14,21]. Both the Cas9-P2A-Puro vector used in our loss-of-function screen (Figure 2C), and the Zim3-dCas9 vector that was used to confirm the effect of L3MBTL2 (Figure 2D), are driven by EF1a promoters.…”

“…While such transcriptional repression depends on multiple factors, including the host cell type, the location within the chromatin of the integration site, the length and deoxynucleotide content of the transgene, and the presence or absence of cis-acting splice signals, transcriptional silencing of these transgenes is often caused by the HUSH complex and its cofactors [1,2,10]. We developed transducible transcriptional reporters that are silenced by the HUSH complex in a manner that is independent of the site of integration in the chromatin, and leveraged this system to identify host genes involved in HUSH complex function [14,21]. Position-independence of these HUSH-sensitive reporters has the advantage that cell sorting is not required to isolate the silenced clones from a heterogeneous pool of transductants, as was the case in previous screens [3].…”

DNA virus infections shape transposable elements activity in vitro and in vivo

“…For the Generation of shKAP1 HFF cell lines, 5 x 10 6 HEK 293T cells were seeded in 10-cm dishes in Dulbecco's minimal essential medium (DMEM) (Gibco) supplemented with 10 % fetal calf serum (FCS, Sigma-Aldrich), and 1 % penicillin-streptomycin (PS, Sigma-Aldrich). Utilizing the Lipofectamine 2000 reagent (Invitrogen), the cells were co-transfected on the next day with packaging plasmids pLP-VSVg (26) and pCMVdeltaR8.9 (kindly provided by T. Gramberg) together either with the plasmid pAPM-D4-miR30-L1221 (a gift from Jeremy Luban; Addgene #115846) (27) expressing a control shRNA or plasmid pAPM-D4 miR30-TRIM28 ts3 (Addgene #115864) (27) encoding for a shRNA targeting KAP1. 48 hours post transfection, the supernatant was harvested, filtered through a 0.45-μm filter and used for transduction of HFF cells.…”