HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration

• Published in: Nucleic acids research Vol. 52; no. 18; pp. 11060 - 11082
• Main Authors: Jang, Sooin, Bedwell, Gregory J, Singh, Satya P, Yu, Hyun Jae, Arnarson, Bjarki, Singh, Parmit K, Radhakrishnan, Rajalingam, Douglas, AidanDarian W, Ingram, Zachary M, Freniere, Christian, Akkermans, Onno, Sarafianos, Stefan G, Ambrose, Zandrea, Xiong, Yong, Anekal, Praju V, Montero Llopis, Paula, KewalRamani, Vineet N, Francis, Ashwanth C, Engelman, Alan N
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 14.10.2024
• Subjects: Capsid - chemistry; Capsid - metabolism; Cell Nucleus - metabolism; DNA, Viral - genetics; DNA, Viral - metabolism; HEK293 Cells; HeLa Cells; HIV Infections - metabolism; HIV Infections - virology; HIV-1 - genetics; HIV-1 - metabolism; Humans; Molecular and Structural Biology; mRNA Cleavage and Polyadenylation Factors - chemistry; mRNA Cleavage and Polyadenylation Factors - genetics; mRNA Cleavage and Polyadenylation Factors - metabolism; Phase Separation; Protein Binding; Protein Domains; Virus Integration
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkae769

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.