AbstractsBiology & Animal Science

My project involves investigating the mechanisms used to selectively package primer tRNALys3 during HIV-1 assembly, place it onto the primer binding site(PBS) on the viral RNA genome, and allow this tRNA to initiate reverse transcription. My basic experimental approach has been to transfect COS7 cells with wild type or mutant HVI-1 proviral DNA and determine the effect of these mutations upon the above processes. I have shown that in the mature virion, there are approximately 8 molecules of tRNALys3, and 12 molecules tRNALys1,2, and that the viral tRNALys3/tRNALys1,2 ratio reflects the cytoplasmic ratio. For example, when COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNALys3 gene, the tRNALys3 content per virion (i.e., per 2 molecules genomic RNA) increases from 8 to 17 molecules, with a corresponding decrease in tRNALys1,2, to 3 molecules/virion. Prl60gag-pol is required for tRNALys3 packaging, and the RT sequences within this molecule probably contain a binding site for this tRNA. In this work, I have also provided evidence that nucleocapsid(NCp7) sequences within Pr55gag contribute to this binding site. This work indicates that a Prl60gag-pol/Pr55gag complex may be responsible for binding tRNALys3 and carrying it into the virion. We next showed that the genomic placement of tRNALys3 onto the PBS occurs independently of precursor proteolysis, but is inhibited by mutations in the basic amino acid sequences flanking the first Cys-His box in NCp7, mutations which do not inhibit packaging of either tRNA Lys3 or Pr160gag-pol into Pr55gag particles. This indicates a role for one or both of protein precursors in the placement. A U5 stem/A-rich loop structure just upstream of the PBS has been implicated in causing the short initiation complexes seen in the mature virion, and causing reverse transcription to move from a low to a high processivity. In agreement with this, we found that the deletion of the A-rich loop immediately upstream of the PBS, while not affecting tRNALys3 placement, resulted in the synthesis of longer elongation products using low RT processivity, with a corresponding decrease in the presence of unextended and 2 base extended tRNALys3 Four A's in the A-rich loop have been implicated in interacting with the USUU sequence in the anticodon loop of tRNALys3 . (Abstract shortened by UMI.)