Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here, we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, providing insights for the development of antiretroviral therapies, such as lenacapavir. IMPORTANCE: Human immunodeficiency virus (HIV) encodes a protein that forms a conical shell, called a capsid, that surrounds its genome. The capsid has been shown to protect the viral genome from innate immune sensors in the cell, to help transport the genome toward and into the nucleus, to keep the components of reverse transcription together for conversion of the RNA genome into DNA, and to target viral DNA integration into specific regions of the host genome. In this study, we show that HIV hijacks two host proteins to bind to capsid sequentially in order to choreograph the precise order and timing of these virus replication steps. Disruption of binding of these proteins to capsid or their location in the cell leads to defective HIV nuclear import, integration, and infection. Mutations that exist in the capsid protein of HIV in infected individuals may reduce the efficacy of antiretroviral drugs that target capsid.