The C-terminal p6 domain of the HIV-1 Pr55(Gag) precursor is required for specific binding to the genomic RNA
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Khoo, Keith K
Ghossein, Shannon
Seissler, Tanja
Wolff, Philippe
McKinstry, William J
Mak, Johnson
Paillart, Jean-Christophe
Marquet, Roland
Bernacchi, Serena
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Abstract
The Pr55Gag 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 Pr55Gag 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 Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag 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 Pr55Gag 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 Pr55Gag 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 Pr55Gag-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.
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RNA BIOLOGY
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15
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7
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© 2018 Taylor & Francis (Routledge). This is an Accepted Manuscript of an article published by Taylor & Francis in RNA Biology on 04 Aug 2018, available online: https://doi.org/10.1080/15476286.2018.1481696
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Genetics