The C-terminal p6 domain of the HIV-1 Pr55 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. Gagp6 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 Gagp6 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 Gagp6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55(Gag) and Gagp6 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.