Phosphorylation of yellow fever virus NS5 alters methyltransferase activity

AbstractSerine/threonine phosphorylation of the nonstructural protein 5 (NS5) is conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to characterize phosphorylated residues of yellow fever virus (YFV) NS5 expressed in mammalian cells. Multiple different phosphopeptides were detected. Mutational and additional mass spectrometry data implicated serine 56 (S56), a conserved residue near the active site in the NS5 methyltransferase domain, as one of the phosphorylation sites. Methyltransferase activity is required to form a methylated RNA cap structure and for translation of the YFV polyprotein. We show the 2′-O methylation reaction requires the hydroxyl side chain of S56, and replacement with a negative charge inhibits enzymatic activity. Furthermore mutational alteration of S56, S56A or S56D, prevents amplification in a viral replicon system. Collectively our data suggest phosphorylation of NS5 S56 may act to shut down capping in the viral life cycle

Naturally occurring genotype 2b/1a hepatitis C virus in the United States

<p>Abstract</p> <p>Background</p> <p>Hepatitis C Virus (HCV) infected patients are frequently repeatedly exposed to the virus, but very few recombinants between two genotypes have been reported.</p> <p>Findings</p> <p>We describe the discovery of an HCV recombinant using a method developed in a United States clinical lab for HCV genotyping that employs sequencing of both 5' and 3' portions of the HCV genome. Over twelve months, 133 consecutive isolates were analyzed, and a virus from one patient was found with discordant 5' and 3' sequences suggesting it was a genotype 2b/1a recombinant. We ruled out a mixed infection and mapped a recombination point near the NS2/3 cleavage site.</p> <p>Conclusions</p> <p>This unique HCV recombinant virus described shares some features with other recombinant viruses although it is the only reported recombinant of a genotype 2 with a subtype 1a. This recombinant represents a conundrum for current clinical treatment guidelines, including treatment with protease inhibitors. This recombinant is also challenging to detect by the most commonly employed methods of genotyping that are directed primarily at the 5' structural portion of the HCV genome.</p

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition

West Nile virus methyltransferase domain interacts with protein kinase G

Background: The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods: We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results: We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions: PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

West Nile Virus Methyltransferase Domain Interacts with Protein Kinase

Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

Subtype Specific Differences in NS5A Domain II Reveals Involvement of Proline at Position 310 in Cyclosporine Susceptibility of Hepatitis C Virus

Hepatitis C virus (HCV) is susceptible to cyclosporine (CsA) and other cyclophilin (CypA) inhibitors, but the genetic basis of susceptibility is controversial. Whether genetic variation in NS5A alters cell culture susceptibility of HCV to CypA inhibition is unclear. We constructed replicons containing NS5A chimeras from genotypes 1a, 2a and 4a to test how variation in carboxy terminal regions of NS5A altered the genotype 1b CsA susceptibility. All chimeric replicons including genotype 1b Con1LN-wt replicon exhibited some cell culture sensitivity to CsA with genotype 4a being most sensitive and 1a the least. The CypA binding pattern of truncated NS5A genotypes correlated with the susceptibility of these replicons to CsA. The Con1LN-wt replicon showed increased susceptibility towards CsA when proline at position 310P was mutated to either threonine or alanine. Furthermore, a 15 amino acid long peptide fused N terminally to GFP coding sequences confirmed involvement of proline at 310 in CypA binding. Our findings are consistent with CypA acting on multiple prolines outside of the previously identified CypA binding sites. These results suggest multiple specific genetic variants between genotype 1a and 1b in the C-terminus of NS5A alter the CsA susceptibility of replicons, and some variants may oppose the effects of others

HCV NS5A is a protein that is rich in both proline residues and disorder and that associates with cyclophilin A via domain 2.

<p>A) Crystallographic model of domain 1 of NS5A (residues 37–213, PDB 1ZH1), which has a well-defined structure. Interestingly, a similar, but alternate structure for domain 1 with a completely different dimer interface (PDB 3FQM) has also been solved and it is currently unknown whether a single conformation or both best represent the intracellular state. B) Linear representation of NS5A. Current evidence suggests the entire carboxy terminus is disordered, but it has traditionally been studied as seperate domains termed domain 2 and 3 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004428#ppat.1004428-Fernandes1" target="_blank">[11]</a>. Red bars represent diprolines. Plot of NS5A disorder prediction from IUPRED (<a href="http://iupred.enzim.hu/" target="_blank">iupred.enzim.hu/</a>).</p

Cyclophilin A binds a flexible loop in the HIV CA, containing glycine 89 and proline 90 and flanked by prolines.

<p>NMR solution structures (left, 1GWP) and multiple crystallographic models of CA (center, 1MG3, 1E6J, 3P05) highlight the structural variability of the Pro loop that is minimized by Cyclophilin A binding (right, 1AK4, 1M9C, 1M9D). Crystal structures are colored by B-factor (a measure of thermal motion and disorder within the structure) as a proxy of conformational flexibilty (red = disordered, blue = ordered). NB: the Pro loop is resolved in only three of the seven protomers in the crystal structure in the unbound state.</p