Background:  All retroviruses use host cell tRNA molecules to prime reverse trancription (RT) of their viral RNA; HIV-1 uses tRNA^Lys3. Before initiation of RT can begin, the 3' end of tRNA^Lys3 must be annealed to the primer binding site of HIV-1 genomic RNA and the two molecules form a complex structure. During this annealing process, the nucleocapsid protein enhances the unwinding of tertiary structures within both RNA molecules. Packaging of tRNA^Lys3 occurs prior to viral budding, at a time when nucleocapsid is still part of the Pr55^Gag polyprotein. In contrast, Pr55^Gag produces virus-like particles on its own and the Pr55^Gag region located between the C-terminal domain of capsid and the C-terminus of nucleocapsid possesses all of the minimal elements required for virus-like particle formation. We have shown that this N-terminal extended form of nucleocapsid (i.e. minimal Gag [mGag]) has greater affinity for HIV-1 genomic RNA than does nucleocapsid alone. We have now studied the tRNA^Lys3 annealing properties of mGag in comparison to those of nucleocapsid.

Methods:  In vitro synthesized viral RNA probes, competitive RNA and human placental tRNA^Lys3 were employed. Protein purification was performed under denaturing conditions by affinity with nickel agarose followed by anion exchange chromatography. RT elongation assays were used to determine the efficiency of tRNA^Lys3 annealing of the different proteins.

Results:  In the absence of competitor, mGag was 5- to 10-fold superior to nucleocapsid in ability to anneal tRNA^Lys3 onto the primer binding site. Furthermore, mGag annealed tRNA^Lys3 onto the primer binding site even in the presence of 4-fold competitor RNA, whereas nucleocapsid did not. Futhermore, in the presence of competitor, our data show that the multimerization ability of mGag can abrogate RT ability. Surprisingly, multimerization of mGag did not abrogate the annealing process but rather resulted in reduced levels of RT processivity. These results were further confirmed using a mutant mGag (M318A), which is known to abolish capsid dimerization and alter virus-like particles formation. We showed that this mutant mGag retained the increased annealing properties of wild type mGag but was less effective than the latter at reduction of RT activity.

Conclusions:  These results strongly suggest that nucleocapsid in the context of the precursor has greater annealing properties than nucleocapsid alone, and that the initial stages of reverse transcription may be regulated by the multimerization ability of Pr55^Gag polyprotein at times prior to cleavage of nucleocapsid.

Keywords: HIV-1; tRNA annealing; packaging