Conserved determinants of retroviral 5' leader dimerization: Insights into genome recognition.

摘要

Retroviral genome dimerization and packaging appear to be mechanistically coupled and mediated by conserved elements within the 5' leader RNA. The dimerization of the HIV-1 genome is promoted by long-range base pairing between an upstream unique-5' element (U5) and residues overlapping the gag start codon (AUG). This U5:AUG complementarity is conserved among divergent retroviruses, suggesting a conserved function. However, previous studies with native and mutant fragments of the HIV-2 5' leader indicated that U5:AUG base pairing inhibits, rather than promotes, dimerization. To better understand the determinants of retroviral genome dimerization, we prepared RNAs corresponding to native and mutant forms of the 5' leaders of HIV-1 (NL4-3 strain), two divergent strains of SIV (cpz-TAN1 and -US strains), and HIV-2 (ROD strain). Subsequently, we probed for the roles of the U5:AUG interaction, the AUG region, the dimerization initiation site (DIS), and the nucleocapsid (NC) protein on 5' leader dimerization. Our findings indicated that the full-length SIVcpz and HIV-2 leader RNAs are more kinetically labile than that of HIV-1. Interestingly, SIVcpz and HIV-2 RNA constructs that favor U5:AUG formation promote 5' leader dimerization, as observed in HIV-1. NC binding to the leader also promotes dimerization in both SIVcpz and HIV-2, but does not induce the formation of non-labile dimers, as observed in HIV-1. Overall, these results suggest that the 5' leader dimerization mechanism is conserved among HIV and SIV, despite the difference in their dimeric lability.;In addition, we investigated the effect of the 5' cap on HIV and SIV 5' leader dimerization. Each viral 5' leader RNA was capped with 7-methylguanosine (m 7G) using the Vaccinia capping system. Our initial results indicated that HIV and SIV leader RNAs with the 5' cap stabilize the monomeric conformation relative to the respective uncapped 5' leader. Hence, we propose that the 5' cap may introduce a structural rearrangement, which inhibits the 5' leader dimerization of HIV and SIV.