PMC

Western blot analysis of phosphorylated ERK levels during inoculation with UV-inactivated HCMV. FFs were inoculated with UV-inactivated HCMV (UV), mock infected (M), or HCMV infected (V). Cell lysates were harvested at the indicated times after infection, and Western blot analysis was performed as described in the legend to Fig. .

³²P pulse-chase analysis of phosphorylated ERK and MEK. (A) Maintenance of phosphorylated ERK during infection. ERK2 was immunoprecipitated from ³²P-labeled, mock (M)- or virus (V)-infected cell lysates after the indicated chase periods and resolved on a 10% polyacrylamide gel. ³²P-labeled ERK2 was detected by autoradiography. (B) Phosphorylated levels of MEK during infection. MEK1 was immunoprecipitated from ³²P-labeled, mock (M)- or virus (V)-infected cell lysates after the indicated chase periods and resolved on a 10% polyacrylamide gel. ³²P-labeled MEK1 was detected by autoradiography. (C) Quantitation of ³²P-labeled ERK and MEK during infection. Stabilization of ³²P-labeled ERK and MEK is a measure of phosphorylated ERK and MEK remaining in virus-infected samples versus mock-infected samples after the indicated chase times. It is defined as the amount of ³²P-labeled ERK or MEK in virus-infected samples divided by that in mock-infected samples. Quantitation of the gels in panels A and B was performed by phosphorimager analysis.

Western blot analysis of phosphorylated ERK levels during HCMV infection. (A) ERK levels early during infection. Total protein from either mock (M)- or virus (V)-infected cells was harvested at the indicated times, separated on 12.5% low-cross-linking polyacrylamide gels, and transferred to Immobilon. Phosphorylated ERK (p-ERK1 and p-ERK2) and nonphosphorylated ERK (ERK1 and ERK2) were detected with antibody directed against ERK1. Time postinfection is indicated above the blot. (B) ERK levels late during infection. Total protein from mock- or virus-infected cells was harvested at the indicated times postinfection and analyzed by Western blotting as described above. “Starved” indicates a lysate from cells that were grown to confluence and serum starved for 24 h. Time postinfection is indicated above the blot.

RSK1 activity during HCMV infection. RSK1 activity was determined by an immune complex kinase assay. RSK1 was immunoprecipitated from serum-starved cell lysates and mock- or virus-infected cell lysates at the indicated times postinfection. RSK1 activity was detected by using an S6 kinase assay kit (Upstate Biotechnology) and [γ-³²P]ATP. Bars represent the average of two independent assays depicted as counts per minute of ³²P-labeled peptide bound to filters as quantitated by scintillation counting; error bars represent half of the range of the two values.

Western blot analysis of phosphorylated ERK levels during HCMV infection of serum-starved cells. (A) FFs were grown to confluence, starved in medium without serum for 24 h, and then infected with virus which had been pelleted and resuspended in medium without serum. Mock infections were performed with serum-free medium containing equivalent concentrations of DMSO as virus-infected samples. Lysates were prepared at the indicated time points and subjected to Western blot analysis as described in the legend to Fig. . (B) Serum-starved FFs were mock or virus infected in the absence of DMSO. Western blot analysis was performed as described above.

ERK activity during HCMV infection, determined by an immune complex kinase assay. ERK was immunoprecipitated from starved cells (Starved) or from lysates at the indicated times after mock (M) or viral (V) infection. After incubation of immunoprecipitated ERK with [γ-³²P]ATP and MBP, the reaction mixtures were run on a 15% polyacrylamide gel and the products were transferred to Immobilon. (A) Autoradiography of ³²P-labeled MBP. The bottom portion of the blot (below 30 kDa) was exposed to film to detect ³²P-labeled MBP. The bracket indicates MBP and breakdown products. (B) Western blot analysis of immunoprecipitated ERK. The top portion of the blot was treated with antibody to ERK1 followed by protein A/G-HRP secondary antibody (Pierce) and detection by chemiluminesence. The antibody to ERK2 (C-14) preferentially precipitates ERK2, which can be detected with the antibody to ERK1 (C-16). Immunoprecipitated ERK1 can be detected on longer exposures. (C) Quantitation of ERK activity in mock- and virus-infected cells. Kinase activity stabilization is a measure of ERK activity in virus-infected cells versus mock-infected cells at a specific time after infection. It is defined as the amount of radioactivity incorporated into MBP in an immune complex kinase assay from virus-infected samples divided by that in mock-infected samples. The values were determined by phosphorimager analysis of the blot from panel A.

Effect of inhibition of the ERK pathway on early viral gene expression. (A) Western blot analysis of UL112-113 protein levels during wild-type HCMV infection in the presence of PD98059. FFs were grown to confluence and serum starved for 24 h. After a 1-h pretreatment with the indicated concentrations of PD98059, the cells were serum stimulated and infected. Cell lysates were prepared 8 h after infection and subjected to Western blot analysis. The UL112-113 43-kDa protein (indicated on the right) was detected by using antibody BSA 2-9 followed by HRP-conjugated secondary antibody and chemiluminescence. (B) Western blot analysis of IE86 and IE72 protein levels during wild-type HCMV infection in the presence of PD98059. The blot from panel A was stripped and probed with antibody CH16.0 followed by HRP-conjugated secondary antibody and detection by chemiluminescence. IE86 and IE72 are indicated by the arrows on the right. (C) Western blot analysis of phosphorylated ERK levels during wild-type HCMV infection in the presence of PD98059. Lysates from the experiment described for panel A were electrophoresed on 12.5% low-cross-linking polyacrylamide gels and subjected to Western blotting as described in the legend to Fig. A. Phosphorylated (p-ERK1 and p-ERK2) and nonphosphorylated forms of ERK1 and ERK2 are indicated on the right. (D) Analysis of CAT protein expression from the UL112-113 promoter during infection with v358-CAT in the presence of PD98059. FFs were grown to confluence and serum starved for 24 h. After a 1-h pretreatment with the indicated concentrations of PD98059, the cells were serum stimulated and infected with the HCMV recombinant v358-CAT. Lysates were prepared 8 h postinfection and assayed for CAT activity. Solid bars represent the average of two independent infections; error bars represent half of the range of the two values.