The Packaging of 7SL RNA by HIV-1.

Abstract

HIV-­1 packages several small cellular RNAs in addition to its genome. Prominent among these is the host noncoding RNA 7SL, of which 12-­16 molecules are found per HIV-­1 particle. This dissertation examined the packaging of 7SL RNA by HIV-­1 to elucidate the cis-­‐ and trans-­acting factors that influence 7SL RNA packaging. 7SL consists of two domains, the Alu domain and the S domain. To determine the trans-­acting packaging factors, minimal virus-­like particles were examined for 7SL packaging. It was determined that minimal ΔNC VLPs retain 7SL RNA primarily as an endoribonucleolytic fragment, referred to as 7SL remnant (7SLrem). Nuclease mapping revealed that 7SLrem is a 111-­nucleotide internal portion of 7SL, with 5' and 3' ends corresponding to unpaired loops in the 7SL two-­dimensional structure. 7SLrem was found in minimal VLPs that contained all Gag proteins except NC, and in minimal VLPs that lacked all of Gag except the C-terminal domain of CA and sp1, indicating that NC protects the Alu domain from processing and that CA/sp1 is sufficient for the acquisition of 7SL. The cis-­acting regions of 7SL that mediate packaging by HIV-­1 were examined by determining the packaging phenotype of 7SL derivatives. Both the Alu domain and the S domain were packaged specifically when expressed separately as truncations of 7SL. However, while the Alu domain competed with endogenous 7SL for packaging in proportion to Gag, the S domain was packaged additively, implying that the Alu domain is packaged by the same mechanism as endogenous 7SL but that the Alu and S domains are packaged via separate mechanisms. These data suggest that multiple domains of 7SL (Alu and S) interact with multiple domains of Gag (CA and NC). The ability of 7SL to interact with multiple domains of Gag suggests that 7SL may play a role in retroviral assembly, possibly by nucleating functional subsets of Gag or by chaperoning Gag to sites of assembly. In addition to the implications for retroviral replication, these data demonstrate the ability to manipulate the cellular RNA content of retroviral virions, which has potential application as a delivery mechanism for modified RNAs such as aptamers.