NP220 mediates silencing of unintegrated retroviral DNA

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.