NP220 mediates silencing of unintegrated retroviral DNA

Abstract

The entry of foreign DNA into many mammalian cell types triggers the innate immune system, a complex set of responses directed at preventing infection by pathogens. One aspect of the response is the potent epigenetic silencing of incoming viral DNAs 1 , including the extrachromosomal DNAs 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 observed after integration 2 – 5 . The factors responsible for this poor expression have not yet been identified. To explore the mechanisms responsible for repression of unintegrated viral DNAs, we performed a genome-wide CRISPR-Cas9 screen for genes required for silencing an integrase-deficient MLV-GFP reporter virus. Our screen identified a DNA-binding protein, NP220; the three proteins of the HUSH complex (MPP8, TASOR, PPHLN1), which silences proviruses in heterochromatin 6 and retrotransposons 7 , 8 ; histone methyltransferase SETDB1; and other host factors that are required for silencing. Further tests by chromatin immunoprecipitation (ChIP) showed that NP220 is the key protein that recruits HUSH, SETBD1, and histone deacetylases HDAC1 and HDAC4 to silence the unintegrated retroviral DNA. Knockout of NP220 accelerates the replication of retroviruses. These experiments have revealed the molecular machinery utilized for the silencing of extrachromosomal retroviral DNA.

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

“…Among our top hits were matrin-type zinc finger proteins ZNF318 and ZNF638 (NP220). Although NP220 is known to recruit HUSH to unintegrated murine retroviral DNA (Zhu et al, 2018), our data predict additional roles for this interaction in the absence of infection. We also detected RPRD2, a regulator of RNA Polymerase II previously identified alongside TASOR as a repressor of LINE-1s and HIV (Liu et al, 2011).…”

“…Whether HUSH has H3K9me3-independent modes of recruitment to target sequences also remains to be resolved. Silencing of unintegrated murine retroviral DNA by HUSH requires NP220 (Zhu et al, 2018), a DNA-and RNA-binding protein thought to be at least partly sequence-specific for cytidine clusters (Inagaki et al, 1996). We find that NP220 and another matrin-type ZNF (ZNF318) interact with TASOR in the absence of virus, raising the possibility that multiple specific adaptors could recruit HUSH in different contexts.…”

“…As well as targeting integrating lentiviruses, HUSH targets full-length transcriptionally-active retrotransposons including LINE-1s Robbez-Masson et al, 2018), and celltype specific genes such as zinc finger transcription factors (ZNFs) Tchasovnikarova et al, 2015Tchasovnikarova et al, , 2017. HUSH is also recruited, via the DNA-binding protein NP220, to repress expression of unintegrated murine leukemia virus (Zhu et al, 2018). The critical role of HUSH in antiretroviral immunity is highlighted by findings that primate lentiviral accessory proteins Vpr and Vpx target HUSH complex proteins for proteasome-mediated degradation (Chougui et al, 2018;Greenwood et al, 2019;Yurkovetskiy et al, 2018).…”

Robust HIV-1 replication in the absence of integrase function

“…As noted above, it has previously been reported that the epigenetic silencing of unintegrated MLV proviral DNA is mediated by the host cell DNA binding protein NP220 acting in concert with the HUSH complex (18). Surprisingly, however, this report also documented that the HUSH complex was not involved in silencing unintegrated HIV-1 DNA, a result that is consistent with the inability of the HIV-2 Vpx protein, a known inhibitor of HUSH complex function, to rescue gene expression from unintegrated HIV-1 DNA ( Fig.…”

“…It is well established that unintegrated retroviral DNA is rapidly loaded with histones that are then decorated with repressive chromatin marks that induce epigenetic silencing (16,17). In the case of unintegrated murine leukemia virus (MLV) DNA, epigenetic silencing is mediated not by PML-NBs but rather by a DNA binding protein called NP220 acting in concert with the human silencing hub (HUSH) complex (18). However, the HUSH complex was reported to not play a role in silencing unintegrated HIV-1 DNA.…”