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.