FIG 1.

HCMV pp71 is protein S-nitrosylated during infection. (A) NuFF-1 cells were infected at a multiplicity of 3 IU/cell with TB40/E-mCherry-UL99eGFP virus; 96 hpi, total lysate was isolated and subjected to a biotin switch assay. Trypsinized proteins were analyzed by mass spectrometry to identify viral peptides that contain a cysteine conjugated to biotin, thereby reveling the site of protein S-nitrosylation. Peptides from 13 viral proteins are shown with the identified sequences. Lowercase “c” denotes the identified biotinylated cysteine. Xcorr*, cross correlation, a measure of the goodness of fit of experimental peptide fragments to theoretical spectra created from the sequence b and y ions; PSM**, frequency of peptide detection; ***, due to partial trypsin digestion, two of the three UL44 peptides and both of the UL25 peptides represent the same identified S-nitrosylated cysteine. Results are from three independent biological replicates. (B) A representative MS/MS spectrum showing the mass shift for the pp71 peptide containing the LxCxD domain. (C) NuFF-1 cells were infected at a multiplicity of 1.0 IU/cell with TB40/E-mCherry-UL99eGFP, and cells were harvested at 96 hpi. After the biotin switch assay, lysates were affinity purified (AP) with avidin beads (top) or immunoprecipitated by pp71-specific antibody (bottom). Purified lysates were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were detected using a pp71-specific antibody (top) or antibody against biotin (bottom). n = 3; representative blots are shown. (D) Specificity of the biotin switch reaction was assessed by performing the reaction in the absence of added biotin. NuFF-1 cells were infected at a multiplicity of 3 IU/cell with TB40/E-mCherry-UL99eGFP virus; 96 hpi, total lysate was isolated and subjected to a biotin switch assay in the presence or absence of biotin. Lysates were affinity purified with avidin beads, and then purified lysates were separated by 8% SDS-PAGE and blotted to nitrocellulose membrane (top). A fraction of the lysates (4%) was run in parallel to confirm protein expression (bottom). Proteins were detected by using specific antibodies. A representative blot from three biological repeats is shown.