An RNA Lariat Intermediate in HIV-1 cDNA Synthesis
David Camerini, University of California, Irvine
The goal of this application is to improve our understanding of the mechanism of HIV-1 cDNA synthesis. We will use tissue culture and in vitro experiments to test the hypothesis that the HIV-1 RNA genome forms a lariat structure at an early stage in minus-strand cDNA synthesis. This novel structure would bring the ends of the genome in close proximity and thereby facilitate the minus-strand template switch. Recent work suggested that the minus-strand template switch in the yeast retroelement Ty1 is accomplished via an RNA lariat intermediate. The lariat intermediate was hypothesized to be formed by a 2'-5' phosphodiester bond between the 5' end of the genomic RNA and the first nucleotide in the R region of the 3' LTR, although the existence of this Ty1 intermediate remains controversial. Our preliminary data indicate that HIV-1 utilizes a similar RNA lariat intermediate during minus-strand transfer since RNAi mediated suppression of the host RNA lariat de-branching enzyme (DBR1) specifically inhibits HIV-1 replication during reverse transcription and allows detection of the lariat intermediate RNase-H assay of RNA topology and by the absence of free 5' ends of HIV-1 genomic RNA. These findings have important implications for HIV-1 replication and for the development of new therapeutics for AIDS. We have the following specific aims:
Aim 1. Further characterize the HIV-1 lariat replication intermediate from DBR1 siRNA transfected and virus infected cells using replication competent HIV-1 and HIV-1 vectors.
Aim 2. Define the sequence requirements for HIV-1 genomic RNA lariat formation using 5' and 3' LTR mutants.
Aim 3. Determine the mechanisms involved in the generation of the HIV-1 genomic RNA lariat replication intermediate.