Integration of viral DNA into the host genome is the hallmark of human immunodeficiency virus 1 (HIV-1) infections. However, in early infection, the most abundant viral DNA species are extrachromosomal unintegrated viral genomes, which despite being competent for gene expression, are poorly expressed in the host cell. This suggests the existence of a restriction mechanism acting selectively on extrachromosomal DNA species. Importantly, restriction of gene expression is relieved upon delivery of the HIV-1 accessory protein Vpr, which degrades host target proteins via the host CRL4-DCAF1 cullin-RING ubiquitin ligase complex.

To identify the responsible Vpr target, I performed a CRISPR-Cas9 forward genetic screen using a custom designed Vpr target sgRNA library. The screen identified SLF2, a poorly characterised protein which recruits the SMC5/6 complex to sites of DNA damage. I confirmed that SLF2 binds the SMC5/6 complex, and gene knockouts validated a functional role for SLF2 and all core SMC5/6 complex components for restriction of gene expression from unintegrated virus. This was independent of the SLF1-mediated DNA damage recruitment pathway. HIV-1 Vpr therefore selectively degrades SLF2 by exploiting CRL4-DCAF1 activity to antagonise restriction.

Unintegrated viral genomes are rapidly chromatinised by the host cell, forming the basis for epigenetic regulation. I showed that the SMC5/6 complex binds unintegrated viral genomes in an SLF2-dependent manner, suggesting that the complex acts directly on viral chromatin. Concomitantly, I showed that SLF2 induces a loss of activating histone marks on unintegrated virus. The SMC5/6 complex belongs to the structural maintenance of chromosomes (SMC) family of proteins that share the ability to topologically entrap and translocate DNA, which can lead to chromatin compaction. Using ATAC-seq, I showed that delivery of Vpr protein or loss of SLF2 increased chromatin accessibility suggesting a loss of compaction. I propose a model in which the SMC5/6 complex is recruited via SLF2 to unintegrated viral genomes, causing chromatin compaction with ensuing silencing of gene expression. Expression of hepatitis B virus, another extrachromosomal DNA virus, is also restricted by the SMC5/6 complex. This suggests that the SMC5/6-mediated pathway of extrachromosomal DNA silencing may have wide-ranging importance for understanding HIV-1 as well as other viral and cellular extrachromosomal DNAs.