Hepatitis C virus RNA replication is regulated by Ras-Erk signalling.
The hepatitis C virus NS5A protein has been previously demonstrated to partially attenuate activation of the Ras-Erk signalling pathway, via a conserved class II polyproline motif located towards the C terminus of the protein. However, the role of Ras-Erk signalling in the virus life cycle remains undetermined. To investigate this, levels of RNA replication were measured in genotypes 1 and 2 transient luciferase subgenomic replicon systems in the context of either pharmacological or genetic (dominant-negative) inhibition of MEK1, a kinase in the Ras-Erk signalling cascade. Incubation in the presence of two inhibitors (U0126 and PD184352) resulted in a decrease in the levels of RNA replication, conversely incubation with inhibitor PD98059 resulted in a modest increase in replication. The results obtained with PD98059 could not be explained by an off-target effect on Cox-2, stability of replicon RNA or stimulation of global translation levels, suggesting stimulation by a yet uncharacterized mechanism. To verify data obtained using pharmacological inhibitors the transient replicon RNA was co-electroporated with a dominant-negative mutant of MEK1. This resulted in a reduction in replication, confirming data seen with U0126 and PD184352. Our data are consistent with the hypothesis that a low level Ras-Erk signalling activity is required for RNA replication. However, complete inhibition of Ras-Erk signalling is inhibitory. These results suggest that perturbation of this signalling pathway by NS5A may be a mechanism to regulate levels of genomic RNA replication.
Gretton
Sarah
S
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Hughes
Mair
M
Harris
Mark
M
D001549:Benzamides / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D016174:Hepacivirus / Q000502:physiology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis*; D015398:Signal Transduction; D014779:Virus Replication; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides; D002070:Butadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D012367:RNA, Viral; C113580:U 0126; D020703:ras GTPase-Activating Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one