Person:

Cherepanov, Peter

Selection of a suitable chromatin environment during retroviral integration is a tightly regulated process. Most retroviruses, including spumaretroviruses, require mitosis for nuclear entry. However, whether intrinsic chromatin dynamics during mitosis modulates retroviral genome invasion is unknown. Previous work uncovered critical interactions of prototype foamy virus (PFV) Gag with nucleosomes via a highly conserved arginine anchor residue. Yet, the regulation of Gag-chromatin interaction and its functional consequences for spumaretrovirus biology remain obscure. Here, we investigated the kinetics of chromatin binding by Gag during mitosis and proviral integration in synchronized cells. We showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity, and redistributed viral integration sites to markers associated with late replication timing of chromosomes. Mutant Gag proteins were, moreover, defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed chromatin. These data indicate that the chromatin landscape during Gag-nucleosome interactions is important for PFV integration site selection and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation.

Nuclear speckles harbor proteins important for transcription and pre-mRNA splicing. HIV-1 preintegration complexes localize to speckles in a capsid (CA)-CPSF6 dependent manner and preferentially target speckle-associated domains (SPADs) for integration. Because the CPSF6-CA interaction is also crucial for integration into gene-dense regions and spliced genes, we correlated the extents of SPAD targeting with gene-dense regions and spliced genes. Our bioinformatic analysis mapped SPADs to gene-dense regions. While the average gene density of the human genome is 9 genes/Mb, nearly 98% of SPADs mapped to regions with gene density > 9 genes/Mb. Our analysis revealed that human chr17 and 19 contained the highest number of genes, intron-containing genes, SPADs, pol II sites, and H3K36me3 sites per Mb. Analysis of HIV-1 integration sites revealed enrichment on chr17 and 19 in wildtype but not in CPSF6 knockout cells. Sites of LEDGF/p75 occupancy and lamina-associated domains were not enriched on human chr17 and 19. Non-primate lentiviruses EIAV, FIV, BIV only marginally targeted SPADs for integration in human cells. As expected, these viruses did not preferentially target gene-dense human chr17 and 19. We next asked whether these lentiviruses targeted gene-dense regions in their natural hosts. As control, the primate lentivirus SIV preferentially targeted gene-dense chr17 and 19 in human cells and gene-dense chr16 and 19 in rhesus cells. Further, the ovine lentivirus MVV did not preferentially target human chr17 and 19 but targeted gene-dense chr11 in the sheep genome. Plausibly, species-specific host factors help guide primate versus non-primate lentiviruses to target gene-dense chromosomal regions for integration. We are currently determining non-primate lentiviral integration sites in additional native host cells. We next asked whether MVV targets spliced genes in sheep. We determined a high correlation (R2=0.6) between integration per kb and the number of sheep introns. By contrast, this correlation was low (R2=0.2) for MVV in human cells due to the lack of SPAD targeting. SPAD-associated human genes contained 0.4 introns per kb, whereas SPAD non-associated genes contained 0.1 introns per kb (P<2E-16), suggesting that SPAD- associated genes are highly spliced. These data suggest a conserved role for pre-mRNA splicing in both primate and non-primate lentiviral replication and indicates that the viruses utilize species-specific factors for trafficking to their preferred integration sites. Acknowledgment: Harvard University Center for AIDS Research (HU CFAR NIH/NIAID fund 5P30AI060354-17).