HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway, Integration Targeting and Replication Efficiency

Abstract

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. During infection HIV-1 enters the nucleus by crossing the nuclear membrane and incorporating itself into the host DNA by a process called integration. Here we show that the viral capsid protein gets tethered to a cyclophilin protein called Nup358, a component of the nuclear membrane gateways that allow transport between the cytoplasm and the nucleus. Altering the capsid protein so that it cannot use Nup358 prevents viral replication in macrophages, a natural target cell type for HIV-1. Intriguingly, these viral mutants are not less infectious in certain immortalised cell lines suggesting that in these cells nuclear entry is regulated differently. In this case similar to wild type virus, the mutant viruses integrate into host chromosomes but they integrate into different regions suggesting that the pathway into the nucleus dictates where the virus ends up in the host chromatin. We also show that another cyclophilin, the cytoplasmic protein cyclophilin A, influences the engagement of Nup358 as well as other proteins involved in HIV-1 nuclear entry. We hypothesise that HIV-1 has evolved to use cyclophilins so that it can access a particular pathway into the nucleus because alternative pathways lead to defects in integration targeting and viral replication in human macrophages.