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Figure 1 | Virology Journal

Figure 1

From: West Nile virus methyltransferase domain interacts with protein kinase G

Figure 1

Multiple sites in NS5 are phosphorylated during a WNV infection. HEK293T cells were infected with buffer (Mock) or WNV (MOI = 10), harvested at 24 hpi, and subjected to immunoprecipitation with α-WNV MTase antibody. (a) The elution was subjected to SDS-PAGE analysis. The 100 kD band corresponding to WNV NS5 in infected cells was excised for mass spectrometric analysis. (b) Western blot using α-WNV MTase antibody after immunoprecipitation identified the 100 kD band isolated from the infected cells as WNV NS5. (c) Spectrum of full scan from the MALDI-TOF of the 100 kD band identified the protein sample as WNV NS5. (d) Enlarged spectrum showing the peaks boxed in C. Arrow points to a singly charged monoisotopic peak of a single phosphorylated peptide corresponding to aa 30–44 with a mass of 1745.04 Da, which includes 79.9 Da corresponding to one phosphorylation. (e) Liquid chromatography tandem mass spectrometry (LC-MS/MS)Data of the phosphorylated peptide Glu30-Arg44 showed a phosphorylation on Ser38. The peptide was purified using a C-18 high performance liquid chromatography (HPLC) column before fragmentation. The mass was calculated for post-translational modifications with an addition of 79.9 for each phosphate. Major fragment ions are labeled with their corresponding b (C-terminal) and y (N-terminal) ions and their charge state. (f) The schematic shows the individual serine or threonine residues present in the WNV NS5 phosphopeptides identified by MALDI-TOF mass spectrometry. Ser38 and Thr451 (in red) were phosphorylated by PKG in vitro. Residues shown in black were present in phosphopeptides with a single serine or threonine. Phosphopeptides with one phosphosite, but more than one serine or threonine residue that may be phosphorylated, are shown in blue. Further analysis is necessary to determine exactly which serine or threonine in these phosphopeptides is phosphorylated. Asterisks indicate the presence of Tyr residues in the phosphopeptide.

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