Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase

Hernández, Sergio; Figueroa, Daniella; Correa, Simón; Diaz, Ariel; Aguayo, Daniel; Villanueva, Rodrigo A.

doi:10.1016/j.bbrc.2015.08.082

Cited by 7 publications

(1 citation statement)

References 26 publications

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“…S29 and S282 residues have been previously described as important for both RDRP activity and viral replication. S29 has already been described previously as substrate of other important kinases in the HCV replicative cycle (Han et al, 2014;Hernandez et al, 2015). Furthermore, S29 is the homologous position of T33 of norovirus, which is also phosphorylated by Akt (Eden et al, 2011).…”

Section: Discussionmentioning

confidence: 71%

Akt Phosphorylation of Hepatitis C Virus NS5B Regulates Polymerase Activity and Hepatitis C Virus Infection

et al. 2021

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Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

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Section: Discussionmentioning

confidence: 71%