Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.

HIV-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors (PRRs). We hypothesized that, if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors, then manipulation of specific interactions between HIV-1 capsid (CA) and host factors that putatively regulate these processes should trigger PRRs and stimulate type 1 interferon secretion. Here we show that HIV-1 CA mutants N74D and P90A, which are impaired for interaction with cofactors Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA) respectively(1-2), cannot replicate in primary human monocyte derived macrophages (MDM) because they trigger innate sensors leading to nuclear translocation of NFκB and IRF3, the production of soluble type-1 interferon (IFN) and induction of an antiviral state. Depletion of CPSF6 with shRNA expression allows wild type virus to trigger innate sensors and interferon production. In each case, suppressed replication is rescued by IFN-receptor blockade demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration suggesting that a viral reverse transcription product comprises the HIV-1 pathogen associated molecular pattern (PAMP). Finally, we show that we can pharmacologically induce wild type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to utilize CPSF6 and cyclophilins to cloak its replication allowing evasion of innate immune sensors and induction of a cell autonomous innate immune response in primary human macrophages (Extended Data Fig 1).

The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.