The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA.

The Pr55^Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55^Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55^Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55^Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55^Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55^Gag by systematically comparing binding of Pr55^Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55^Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55^Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~ 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55^Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation. [ABSTRACT FROM AUTHOR]