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).

SUMMARY Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyses trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3 but the significance of this remains unclear. Here, we identify a new class of short RNAs, ~50-200 nucleotides in length, transcribed from the 5′-end of polycomb target genes in primary T-cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.