Background:  Recent work suggests that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate. The lariat facilitates minus-strand cDNA transfer from the 5’ R region to the 3’ R region by formation of a 2’ to 5’ phosphodiester bond between the 5’ end of Ty1 genomic RNA and the first nucleotide of the 3’ R region. We hypothesize that HIV-1 might also form a genomic RNA lariat by this novel mechanism and therefore siRNA-mediated knockdown of the human RNA lariat de-branching enzyme (DBR1) expression would specifically inhibit HIV-1 replication.

Methods:  We designed 3 siRNA molecules targeting DBR1 and used to transfect GHOST-R5X4 cells followed by HIV-1 infection. Cell supernatants were collected 24 hours later for HIV-1 assay by p24 ELISA, and cells were lysed and DNA was isolated to evaluate the synthesis of HIV-1 cDNA by real-time quantitative polymerase chain reaction (PCR). RNA was isolated 2 to 24 hours later and the HIV-1 genomic RNA lariat was detected by 5’ RACE and real-time (RT) PCR. This RNA lariat was also found when cells were infected with integrase-deficient HIV-1. To characterize the putative HIV-1 genomic RNA lariat replication intermediate, we defined the sequence requirements for lariat formation using 5’ and 3’ LTR mutants.

Results:  We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knock-down led to decreases in viral cDNA and protein production. These effects could be reversed by co-transfection of a DBR1 cDNA, indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. We found that DBR1 mRNA suppression inhibited detection of HIV-1 RNA 5’ ends and also allowed detection of the HIV-1 genomic RNA lariat by RT-PCR both in wild type and integrase-deficient HIV-1-infected cells. Moreover, stability of the HIV-1 genomic RNA lariat is affected by the 5’ and 3’ branch-point nucleotides.

Conclusions:  These data indicate that the HIV-1 utilizes an RNA lariat intermediate during minus-strand transfer. This will have profound implications for our understanding of HIV-1 cDNA synthesis.