HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration
Sooin Jang, Gregory J. Bedwell, Satya P. Singh, Hyun Jae Yu, Bjarki Arnarson, Parmit K. Singh, Rajalingam Radhakrishnan, AidanDarian W Douglas, Zachary Ingram, Christian Freniere, Onno Akkermans, Stefan G. Sarafianos, Zandrea Ambrose, Yong Xiong, Praju Vikas Anekal, Paula Montero Llopis, Vineet N. KewalRamani, Ashwanth C. Francis, Alan Engelman
Abstract
HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.