Replication and Evolution of Primate Lentiviruses. - PROJECT SUMMARY Nup358 (RanBP2) is a major component of the nuclear pore complex (NPC). Nup358 comprises four RAN- binding domains (RBD1-4) interspersed among several unique domains. The long filaments that extend from the nuclear pore rim into the cytoplasm are Nup358 multimers, and HIV-1 capsids bind to Nup358 via its C-terminal cyclophilin-homology domain (CHD). Importantly, this interaction is nearly identical to the binding of HIV-1 capsids to cyclophilin A (CypA), which is known to affect sensitivity to other capsid-interacting restriction factors, including restriction factors such as TRIM5a and TRIMCyp. Our comparative analysis of different primate lentiviruses, and capsid interactions with Nup358 homologs from reservoir and spillover hosts, revealed a previously unknown effect of the fourth RAN-binding domain (RBD4) on specificity of the CHD. Thus, the RBD4 and CHD work together as a single functional unit, or didomain. Preliminary experiments indicate that RBD4-dependent interactions between the CHD and capsids affect viral nuclear import and infectivity. The most striking example involves emergence of HIV-2, which had to adapt to differences between the Nup358 homologs of sooty mangabeys (the reservoir host) and humans (the spillover host). Finally, we found that the capsid-CHD interaction is more conserved than previously reported, which is compelling evidence that this interaction contributes to optimal fitness of HIV-1 and related lentiviruses, such as HIV-2 and the simian immunodeficiency viruses (SIVs) of old-world monkeys. For this project, we will employ a powerful strategy that includes comparative analyses of HIV-1, HIV-2 and the other SIVs (and their respective hosts). Our research plan involves complementary use of virological, biochemical and structural (CryoEM/ET) approaches to establish the molecular level details of the interactions between lentivirus capsids and the RBD4-CHD didomain of Nup358, and how this contributes to viral replication. Specifically, we seek to understand how the RBD4-CHD didomain contributes to nuclear docking, nuclear transport and replication of HIV-1 (AIM 1), how the RBD4 governs specificity of capsid interactions with the RBD4-CHD didomain (AIM 2), and what effect these interactions have on overlapping interactions with cyclophilin A (CypA) and TRIM5a (AIM 3).
