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“Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral

RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains Belnacasan cell line both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic

analysis of purified protein indicate that nsP1 has a mixed alpha/beta structure and is in the folded native conformation. (C) 2011 Elsevier Inc. All rights reserved.”
“The human kinome comprises over 800 individual kinases. SU5402 mw These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases

regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target ever effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability.