West Nile virus methyltransferase domain interacts with protein kinase G
© Keating et al.; licensee BioMed Central Ltd. 2013
Received: 19 February 2013
Accepted: 11 July 2013
Published: 22 July 2013
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.
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.
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.
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.
KeywordsFlavivirus Phosphorylation Interaction Protein kinase G
The Flavivirus genus includes a number of medically-relevant arthropod-borne viruses, transmitted primarily by mosquito or tick. Mosquito-borne flaviviruses, such as yellow fever virus (YFV) and dengue viruses (DENV (1–4)), are transmitted primarily by Aedes mosquitoes [1, 2], while West Nile virus (WNV) and Japanese encephalitis virus are transmitted primarily by Culex mosquitoes [3, 4]. Tick-borne flaviviruses, including Langat virus (LGTV), constitute a distinct phylogenetic lineage and are poorly, if ever, transmitted by mosquitoes [5, 6]. The Flaviviridae family, including the Flavivirus genus, consists of single-strand (+)-sense viruses. The genome is translated from a single open reading frame into a polyprotein, which is cleaved into separate structural and non-structural proteins. In flaviviruses, the non-structural protein NS5 is comprised of an N-terminal methyltransferase (MTase) domain [7, 8] and a C-terminal RNA-dependent RNA polymerase (RdRp) domain . The Hepacivirus and Pestivirus genera have NS5A and NS5B proteins; NS5B has RdRp activity .
A number of cellular kinases are able to phosphorylate viral proteins from a wide range of viruses, and the roles of kinases in viral lifecycles continue to be studied . Serine/threonine phosphorylations of NS5 and NS5A are conserved throughout the Flaviviridae family  – phosphorylated NS5/NS5A is found in all three genera and in both mosquito- and tick-borne Flaviviruses [13, 14]. The identity of many kinases able to phosphorylate flaviviral NS5, and the role of these kinases in viral infections, are unknown. We have previously demonstrated mammalian protein kinase G (PKG), a cGMP-dependent serine/threonine kinase, is able to phosphorylate dengue (DENV-2) NS5 at a site conserved among all mosquito-borne flaviviruses (Thr449, in the RdRp domain of NS5) . Furthermore, DENV-2 NS5 is also phosphorylated by mosquito PKG, and alterations in PKG activity changes the flight behavior of Aedes and non-Aedes mosquitoes  raising the possibility that flaviviruses alter their own transmission from vectors.
In mammals, PKGI and PKGII are expressed from separate genes, and PKGI (referred to as PKG in this paper) is alternatively spliced at the extreme N-terminus into PKGIα and PKGIβ isoforms . Both cGMP binding  and autophosphorylation  are involved in structural changes that induce PKG’s catalytic activity, increasing the enzyme’s ability to phosphorylate a substrate. Binding of cGMP to PKGI also induces a solvent-exposed enzymatic conformation that is hypothesized to allow for substrate binding to PKG . Previous results showed that PKGIα, PKGIβ, and PKGII are all able to phosphorylate DENV-2 NS5 . PKGIβ has previously been shown to increase replication of an HIV-1 LTR-reporter , but a specific role for PKG in viral lifecycles has not been demonstrated.
In this paper, we have expanded our study of PKG’s role in flaviviral infections from Aedes-borne DENV to Culex-borne WNV. We identified multiple phosphorylation sites in WNV NS5 and report that a truncated WNV NS5 (MTase only), without the previously identified NS5 phosphorylation site in DENV, is also a substrate for phosphorylation specifically by PKG. The Ser38 site in the MTase domain is phosphorylated during a WNV infection. Additionally, we showed that WNV NS5 is able to form a stable interaction with human PKGIβ. Tick-borne LGTV NS5 did not interact with PKG, suggesting further substrate-specificities of PKG for mosquito- versus tick-borne flaviviral NS5. The MTase domain alone of WNV NS5 interacted with PKG, and mutating specific residues involved in the interaction rendered the WNV replicon non-functional. This is the first demonstration that the αE helix of NS5 is critical for WNV replication and that PKG binding occurs at this critical region, even though PKG itself is likely not required for viral replication.
WNV NS5 is phosphorylated in infected HEK293T cells
The WNV MTase domain is a major substrate for PKG phosphorylation
To identify specific residue(s) phosphorylated by PKG, an in vitro kinase reaction using WNV MTase domain and PKGIα was performed. The reaction was subjected to SDS-PAGE analysis, and the gel band corresponding to WNV MTase (33 kD) was excised and in-gel trypsin-digested. Half of the sample was treated with phosphatase. Both the phosphorylated and dephosphorylated samples were analyzed with MALDI-TOF mass spectrometry. Results from MALDI-TOF analysis identified a specific peptide corresponding to aa Ser38-Lys45 in the MTase domain (Figure 2(b)), which lost ~80 Da in mass (equivalent to a phosphate group) after phosphatase treatment compared to the untreated peptide (Figure 2(c)). These results support our hypothesis that the greater mass of the Ser38-Lys45 peptide in the non-phosphatase-treated sample was due to the presence of a phosphate group on Ser38. Subsequent MS/MS analyses also confirmed that the Ser38 residue of WNV MTase was phosphorylated specifically by PKG in vitro (Figure 2(d)). Subsequent mutational analysis of the 38 residue suggested that WNV was still able to replicate even when it was unphosphorylatable at Ser38 in WNV replication (data not shown), thus we shifted our focus to a more overall role for PKG in WNV infections.
WNV viral production is enhanced in BHK-21 cells expressing PKG
WNV NS5, but not LGTV NS5, interacts with mammalian PKGIβ
The WNV MTase domain can interact with PKG
αE sequence in flaviviruses
Virus (GenBank accession number)
αE sequence (aa187-202)
Residues within the αE helix region of WNV MTase are involved in MTase’s interaction with PKG
αE mutations inhibit WNV replication
Residues within the αΕ region have not been identified as involved in MTase’s N-7 or 2’-O methylation reactions. However, we wanted to ensure that our introduction of mutations was not inhibiting MTase activity to induce the loss of replication. Using a 190 base pair capped RNA as a substrate we compared the N7 and 2’0 MTase activity of recombinant WNV MTase with that of the four αΕ mutant WNV MTase. No clear defect in MTase activity was seen (Figure 7(b)).
While WNV NS5 has been previously shown to be phosphorylated , the specific sites have not been previously mapped. The MTase domain is an effective substrate for PKG phosphorylation, and is phosphorylated specifically at the Ser38 site during a WNV infection and by PKG in vitro. Additionally, WNV NS5 forms a stable interaction with mammalian PKG. Specific charged residues within the αE helix of the MTase domain are able to mediate this interaction, and mutating these same residues prevents WNV replication.
When PKG is present in cells, there is a stable interaction between WNV NS5 and PKG. Stable interactions between a cellular kinase and viral substrate are not unprecedented , and in fact the hepatitis C virus protein NS5A, related to WNV NS5, is able to co-immunoprecipitate with cellular kinase phosphatidylinositol 4-kinase type IIIα from cell culture . It is unknown why this, or any, kinase might have a more stable interaction - it is possible that the kinase’s association with the viral protein has an effect beyond the substrate phosphorylation itself. Additional insight into the role of PKG in flaviviruses may require the development of an NS5 that is both replication competent and unable to interact with PKG.
We have shown that mutating the positive residues in the αE region that are involved in the NS5-PKG interaction prevents replication of the WNV replicon. As multiple mutations were added to the WNV replicon, it is unclear if factors other than WNV NS5-PKG interaction have been disrupted. For example, the literature suggests that several charged residues (mostly Glu) in the αE region are involved in the interaction between DENV-2 NS5 and NS3 . We changed four basic residues in the same region disrupting the MTase-PKG interaction, but possibly also disrupting the NS5-NS3 interaction or other functions. These mutations did not dramatically alter the MTase function of NS5. While we do not yet have a specific mechanism for the role of the NS5-PKG interaction, ourData do show that the αE region is important for WNV replication and that PKG utilizes this important region for its interaction.
Flaviviruses replicate well in BHK-21 cells, which are naturally PKG-deficient , thus it seems that PKG is not necessary for propagation of flaviviruses in cell culture. When phosphorylation of flaviviral NS5 (DENV-2) was first detected by relatively unsensitive techniques, it was particularly prominent only under low serum conditions . However, the NS5 protein from multiple mosquito-borne flaviruses (WNV (above), DENV-2 , and YFV ) have now been shown to be a substrate for PKG, and we show here that PKG’s expression in BHK-21 cells is associated with an enhancement in WNV replication. As flaviviruses have limited coding ability and high replication rates, the conservation suggests an evolutionary advantage for the preservation of PKG substrate sites. Furthermore, activation of PKG in mosquitos leads to increased wing activity , similar to the increase in foraging noted in insects that are not viral disease vectors [28, 29]. This could be the mechanistic basis for the recently reported increase in mosquito flight caused by DENV  and could facilitate viral transmis-sion and account for the conservation of PKG phosphorylatable sites in mosquito-transmitted flaviviruses.
We show that mosquito-borne, but not tick-borne, flaviviral NS5 are substrates for phosphorylation by and interact with mammalian PKG. The data here suggest further substrate specificities for mammalian PKG between mosquito- and tick-borne flaviviral NS5, as LGTV NS5 does not interact with PKG as WNV NS5 does. The WNV MTase domain alone is able to interact with PKG, so this domain is a significant substrate for both PKG phosphorylation and interaction. The αE helical region of the MTase domain is involved in the interaction between WNV NS5 and PKG. Specific mutations within this region that interrupt the WNV MTase-PKG interaction result in non-functional WNV replication. While a specific mechanism for PKG’s phosphorylation of and interaction with NS5 affects flaviviral replication remains unknown, the results provide more information about an overall role for PKG in both Aedes- and Culex-borne flaviviral infections.
Materials and methods
All research was approved by the relevant committees at UW Madison.
Cell culture and viral infections
Human embryonic kidney (HEK293T, ATCC#CRL-11268) and baby hamster kidney cells (BHK-21; ATCC #CCL-10) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Sigma). African green monkey kidney cells (Vero; ATCC #CCL-81) were maintained in minimal essential medium (MEM, Invitrogen, Carlsbad, CA) supplemented with 10% HI-FBS, 2 mML-glutamine and 1.5 g/L sodium bicarbonate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in 5% CO2 at 37°C.
WNV was produced from an infectious cDNA clone of an isolate from New York in 2000 . RNA was in vitro transcribed from the cDNA clone and then electroporated into BHK-21 cells as previously described . Viral titers were determined by plaque assays on Vero cell monolayers. For WNV infections, HEK293T were seeded and incubated at 37°C overnight. Medium was removed, and the cells were inoculated with WNV in DMEM medium supplemented with 1% HI-FBS at 37°C. At 1hour post-inoculation, the medium was replaced with DMEM supplemented with 10% HI-FBS, 50 U/mL penicillin, and 0.05mg/mL streptomycin, and the cells were incubated at 37°C. Supernatants were harvested at the indicated time points post-infection and stored in aliquots for titering by plaque assay on Vero cells. For mass spectrometry analysis, infected HEK293T cells were lysed by homogenization in Tris-Cl buffer containing 1% NP-40, 1X Halt Inhibitor Protease Cocktail (Pierce, Rockford, IL) and 1X Halt Phosphatase Inhibitor Cocktail (Pierce). WNV NS5 was purified from the cell lysate by immunoprecipitation overnight with a custom-made rabbit polyclonal antibody against WNV NS5 (Covance, Princeton, NJ).
Mass spectrometry sample preparation, loading, and analysis
WNV NS5 (full length or MTase domain only) protein samples were prepared for mass spectrometry andData analysis as described previously . In short, WNV NS5 immunoprecipitated protein from HEK293T cells (in case of viral infection) or E. coli-expressed Ni-NTA affinity purified WNV MTase domain was subjected to run in 10% SDS-PAGE gel. After the targeted band was excised from gel (identified with GelCode Blue stain (ThermoFisher Scientific, MA)), in-gel digestion with Trypsin Gold (Promega, WI) in combination with Proase MAX Surfactant (Promega, WI) were performed at 50°C for 90minutes. After peptide recovery from gel pieces one half of the sample was treated with 20 units of calf intestinal alkaline phosphatase (CIAP) for dephosphorylation at 37°C for two hours. Both phosphorylated and dephosphophorylated peptides were then desalted with C18 Zip-Tip column (Millipore, MA) and run in both MALDI-TOF/TOF (Applied Biosystem, CA) and Finnigan LTQ Linear Ion Trap mass spectrophotometer (Thermo-Fisher Scientific, MA) for LC-MS/MS at University of Wisconsin-Madison Mass Spec Facility Center as described previously . MALDI-TOFDatawere collected from the range of m/z 600 and 4000 and the spectra analysis was performed in GPS Explorer software (Applied Biosystem, CA). MascotDatabase (Matrix Science, UK) search andData comparison before and after CIAP treatment, detailed matching of mass with peptide identification was performed in a web-based software in Protein Prospector (v 4.27.1) called MS-Digest tools. ESI precursor ion scan mass spectra were obtained using Finnigan LTQ Linear Ion Trap mass spectrometer equipped with a nanoelectrospray ion source. Zorbax 300SB-C18 nanoflow HPLC column (150 mm × 75 μm, Agilent Technologies, Santa Clara, CA) was eluted with a linear gradient of water-acetonitrile in the presence of 0.1% formic acid at a flow rate of 0.2 μl/min. The remainder of the protocol was followed as described previously  and full scan spectra were recorded from m/z 200–2000 followed by MS/MS spectra in Xcalibur software (ThermoFisher Scientific, MA). MS/MS spectra of detected phosphopeptides were searched against the FASTADatabase using the Sequest search program within BioWorks Rev3.3 software (ThermoFisher Scientific, MA).
DNA constructs and cloning
The full-length WNV NS5 was amplified from a 6XHis-tagged WNV NS5 in pET-LIC template by PCR (forward primer 5′-GCTAGCATGGCCGGTGGGGCAAAAGGACGC-3′ and reverse primer 5′-TTAATTAACAGTACTGTGTCCTCAACCAAAG-3′) and cloned into a 6XHis IRES GFP vector  using NheI and PacI. The WNV MTase domain (aa 1–300) was amplified from the full-length clone using the forward primer 5′-GCTAGCATGGCCGGTGGGGCAAAAGGACGC-3′ and reverse primer 5′-TTAATTAAGTTCTCATCGTGGTGCCACGTCG-3′ and cloned into the IRES GFP vector using NheI and PacI. The RdRp domain (aa 301–906) was amplified to include a 5′ HA tag with the forward primer 5′-ATGTACCCATACGATGTTCCAGATTACGCTCACCCATATAGAACC-3′ and reverse primer 5′-TTAATTAACAGTACTGTGTCCTCAACCAAAG-3′ and cloned into the IRES GFP vector using NheI and PacI. WNV MTase was cloned into the pQE-30 bacterial expression system (Qiagen, Germantown, MD) with BamHI and HindIII for protein expression and purification. Full-length LGTV NS5 (aa 1–904) was cloned into the IRES GFP vector with an HA tag, using NheI and PacI.
C-terminally Flag-tagged human PKGIβ was obtained from Dr. Kuan-TehJeang , and the full-length tagged gene was cloned in the pCI-Neo vector using NotI and XhoI. The resulting PKGIβ-Flag construct was used in DNA transfections.
The pACYC WNV RLuc2A Replicon has been previously described . The αE region was mutated by PCR amplifying the SpeI-XbaI fragment (nucleotide 6771–9975), cloning into the TOPO vector (Invitrogen, Carlsbad, CA), and performing site-directed mutagenesis using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The mutated fragment was then cloned back into the replicon vector.
Expression and purification of proteins
His-tagged WNV MTase was expressed in M15/pREP4 E. coli (Qiagen) using the pQE-30 bacterial expression system. Protein expression was induced by incubating E. coli with 1 mm isopropyl-β-D-thiogalactopyranoside (Invitrogen) for 7 hours. Bacterial cells were collected by centrifugation and lysed by sonication in buffer containing 50 mm Tris (pH8.0), 300 mm NaCl, and 10 mm imidazole. Cleared lysate was incubated with Ni-NTA beads (Qiagen) for His-tag binding, and bound protein was eluted in buffer containing 50 mm Tris (pH8.0), 300 mm NaCl, and 250 mm imidazole.
In vitrokinase assay
Each in vitro kinase reaction was performed by incubating 1 μg of purified WNV NS5 (full-length or MTase domain only) substrate with 100 ng bovine lung PKGIα (Calbiochem, Gibbstown, NJ), 5 μCi of γ-P32-labelled ATP (Perkin Elmer, Waltham, MA), 10 μM cGMP (Calbiochem) and PKG reaction buffer as described previously  in a total reaction volume of 25 μL for 15 minutes at 30°C. The reactions were incubated for 30 minutes at 30°C, then terminated by adding 10 μ L Laemmli buffer and denaturing at 95°C for 3 minutes. Entire samples were separated by 10% SDS-PAGE, and the gel was stained with Coomassie blue, fixed, and dried for autoradiography with exposure on Kodak BioMax XAR film. For mass spectrometric analysis, samples were prepared as described above, except with 200 μM cold ATP in place of the radiolabeled ATP. The samples were incubated at 30°C for 60 minutes before terminating the reaction by boiling and subjecting the samples to 10% SDS-PAGE for subsequent mass spectrometry preparation and analysis.
Establishment and analysis of stable BHK-21 expressing PKG cell line
BHK-21 cells in a 6-well plate were transfected with 1.5 μg of pCI-Neo DNA plasmid expressing human PKGIβ-Flag or empty vector pCI-Neo DNA plasmid to generate BHK + PKG and BHK + EV cells, respectively. Cells were incubated for 48 h in DMEM supplemented with 10% HI-FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Transfected cells were then maintained with 800 μg/μL neomycin (Invitrogen). Cells were lysed by freeze/thaw cycles for further analysis. The expression of PKG was analyzed by Western blot analysis using monoclonal mouse α-Flag (Sigma) and mouse monoclonal α-actin (Calbiochem). PKG activity was confirmed using the CycLex cyclic GMP dependent protein kinase assay kit (MBL International, Woburn, MA) according to the manufacturer’s protocol.
HEK293T cells were transfected with DNA constructs using the TransIT-LT1 transfection system (MirusBio, Madison, WI). Cells in T-75 flasks were transfected with 15 μg of total DNA (co-transfection of NS5 and PKG, NS5 alone, PKG alone) or mock-transfected (no DNA). Cells were harvested by scraping at 48 hours post-transfection and lysed by homogenization in 1mL lysis buffer (50 mMTris-CL (pH8.0), 75 mMNaCl, 75 mMKCl, 4 mM MgCl2, 1% NP-40) containing 1X Halt Protease Inhibitor Cocktail (Pierce). 500 μg of total protein from the lysate was subjected to immunoprecipitation using 10 μg of α-Flag (mouse monoclonal antibody, Sigma) in the presence of Protein G agarose beads (Invitrogen). The beads were boiled in Laemmli sample buffer to elute the precipitated proteins. The precipitated protein samples were separated on 10% SDS-PAGE gels and transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were incubated with the following antibodies: custom-made rabbit polyclonal α-WNV MTase serum to detect WNV NS5 and WNV MTase (1:1000; Covance), α-Flag to detect PKG (mouse monoclonal antibody (1:500, Sigma) or rabbit polyclonal antibody (1:2000, GenScript, Piscataway, NJ)), mouse monoclonal antibody against actin (1:1000; Calbiochem), and α-HA to detect WNV RdRp and LGTV NS5 (custom monoclonal antibody from mouse serum; 1:20, from hybridoma clone 12CA5).
In vitrobinding assay
DNA encoding human PKGIβ in pCI-Neo (or an empty pCI-Neo plasmid for negative control) was used in a TNT reaction with 35S-labeled methionine (Perkin Elmer, Waltham, MA) to produce radiolabeled PKG protein. MTase proteins were expressed in E. coli using the pQE-30His bacterial expression system (Qiagen). The His-tag purification of MTases was done with the Ni-NTA beads as described above, but the protein was not eluted from the beads. Binding reactions consisting of 3 μL Ni-NTA beads (no protein), 10 μL Ni-NTA beads from His-purification procedure (attached MTase protein), and 10 μL TNT-produced sample were assembled and incubated overnight in co-IP lysis buffer (described above). Beads were washed, then boiled to elute bound proteins. The samples were separated by 10% SDS-PAGE. The gels were stained with Coomassie, fixed, dried, and the presence of PKG was visualized by autoradiography.
WNV NS5 replicon mutagenesis, RNA transcription, transfection, and replication analysis
WNV replicon RNA (wild-type and αE mutant WNV replicon) was linearized with XbaI and in vitro transcribed with themMessagemmachine kit (Ambion, Carlsbad, CA) (manufacturer’s protocol). RNA was electroporated into 8 × 106 HEK293T cells and plated in 12-well plates as previously described . The medium was removed and cells were lysed at various time points post-transfection in 250 μL 1X RenillaLysis Buffer (Promega). The lysates were frozen at −80°C prior to the assay, and the total protein in the lysates was quantified with the Coomassie Plus (Bradford) Assay Kit (Pierce). To determine the luciferase activity, 10 μg of total protein from the lysate was added to 90 μL of the Renilla luciferase substrate. Luciferase activity was detected using a Glomax 20/20 Luminometer (Promega) and measured in relative light units (RLUs) per 10 μg of total protein. Triplicate wells were transfected for each sample at each time point.
Capped RNA substrates: Capped RNA substrates for methyltransferase assays were generated as described by Ray et al. . Briefly, capped RNA substrates representing the first 190 nucleotides of the 5′-terminal WNV genome were in vitro transcribed from PCR products generated with primer 1 (5′-CAG TAA TAC GAC TCA CTA TTA GTA GTT CGC CTG TGT GAG CTG ACA AAC TT-3′ and primer 2 (5′-TCT TCA GTC CAA TCA AGG ACA ACA CGC-3′), primer 1 containing a bacteriophage T7 class II ϕ2.5 promoter (italicized) that allows ATP initiated transcript using T7 RNA polymerase . In vitro WNV RNA transcripts were capped in the presence or absence of SAM with (α-32P) G*TP and Vaccinia virus capping enzyme (CellScript, Madison, WI) to generate G*pppA-RNA and m7G*pppA-RNA. The resulting capped RNA was purified through a G-50 column (GE Healthcare, Waukesha, WI) and subsequently used in methylation assays.
Cap methylation assays: The N-7 methylation reaction was performed at 30°C for 15 min in N7 buffer (50 mM MES, pH6.0, 1 mM DTT, 1 mM MgCl2, and 6 mMKCl) containing 5000 CPM G*pppA-WNV RNA, 0.5 mg purified wild-type or αE mutant methyltransferase and 100 mm SAM. The 2′-O methylation reaction was performed at 30°C for 45 minutes in 2′-O buffer (50 mm Tris, pH8, 1 mm DTT, 1 mmmgCl2, and 6mm KCl) containing 5000 CPM m7G*pppA-WNV RNA, 0.5 mg purified wild-type or αE mutant WNV methyltransferase and 100 mM SAM. Control m7GpppA-RNA and m7GpppAm-RNA was prepared using Vaccinia virus capping enzyme or Vaccinia virus Cap 1 methyltransferase (Cellscript, Madison, WI) respectively, following the manufacturers protocol. Methylation reactions were digested for one hour at 68°C with Nuclease P1 (US Biological, Swampscott, MA) and analyzed by polyethyleneimine cellulose thin layer chromatography in 0.2M ammonium sulfate.
A two-tailed unpaired Student’s t test (GraphPad, San Diego, CA) was used to test the differences between two groups (e.g. comparison of the replication of individual αΕ mutant replicon to replication of the wild-type WNV replicon, comparison of PKG activity between two stable cell lines). A two-tailed Mann–Whitney U test was used to test the differences between two groups in WNV infections. A P value ≤ 0.05 was considered statistically significant.
The authors would like to acknowledge Dr. Etti Harms for making genetic constructs, and Dr. Israr-ul Ansari for his technical assistance and critical reading of the manuscript. JAK is funded by the NIH-funded Cellular and Molecular Parasitology Training Program (2T32AI007414). RS is funded by a Veteran’s Association Merit Award.
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