Significance Invading viral DNAs constitute a high risk for the infected cell and are high-profile targets of antiviral host factors. Nevertheless, little is known about the silencing machinery in the nucleus that acts to prevent transcription or retroviral integration of extrachromosomal DNA. We here identified CHAF1A and CHAF1B as two players that mediate silencing of unintegrated HIV-1 DNAs. Our findings provide evidence that these factors act independently of their canonical nucleosome assembly complex to induce silencing early in infection. The characterization of the intrinsic cellular defense mechanism against incoming DNA is relevant to transient gene delivery, as mediated by virus-based vectors in gene therapy.