The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

Abstract

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32P-orthophosphate and 3H-leucine identified a 3.5-fold increase in the 32P:3H counts per minute (cpm) ratio of N in the virion as compared to the 32P:3H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32P:3H cpm ratio of N from the virion was 10.5-fold greater than the 32P:3H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.

Fig. 1

Calf intestinal alkaline phosphatase treatment (CIAP) of the IBV N protein. ³²P-orthophosphate-labeled IBV N protein from lysates of infected Vero cells and from virions was immunoprecipitated with rabbit anti-IBV polyclonal antibody before separating by SDS–PAGE. Panel A represents the autoradiogram and panel B, the Western blot using chicken anti-IBV (Gray) polyclonal antibody. The labeled N protein was treated with 20 U CIAP and resolved by denaturing electrophoresis in a 15% polyacrylamide gel. Panel C represents an autoradiogram of the virion N. Panels D and E show the autoradiogram and Western blot, respectively, of cellular N obtained 8 h p.i.

Fig. 2

Time course of phosphorylation of the IBV N protein in the presence of actinomycin D. Phosphorylation of IBV N protein from IBV-infected Vero cells was evaluated by labeling with 100 μCi ³²P-orthophosphate for 1 h in the presence of 5 μg/ml actinomycin-D. At varying times p.i. (as indicated above the figure), equal amounts of whole cell lysate were resolved by 15% SDS–PAGE and the labeled N visualized by autoradiography. Panel A represents the phosphor image, and panel B represents the Western blot using rabbit anti-N polyclonal antibody. IBV N protein was observed at approximately 48 kDa molecular mass.

Fig. 3

Stability of IBV N protein. Panel A represents a phosphor image, and panel B represents a graphical representation of the densitometric analysis of the bands obtained in the phosphor image. IBV-infected Vero cells were pulse labeled with 25 μCi ³⁵S-methionine for 30 min and chased with a large excess of unlabeled methionine. The N was obtained from IBV-infected cell lysates by immunoprecipitation with rabbit anti-N polyclonal antibody. The 0-h chase time correlated with 8 h p.i. Lysates from uninfected cells precipitated with rabbit anti-N polyclonal antibody (0 h) and lysates from infected cell precipitated with preimmune serum (PS) are shown. Error bars represent standard deviations of two densitometric measurements from one experiment.

Fig. 4

Pulse-chase analysis determined the stability of phosphorylation on the IBV N protein. IBV-infected Vero cells were labeled with ³²P-orthophosphate for 2 h and chased with unlabeled phosphate starting at 8 h p.i. until 11 h p.i. Panel A represents a phosphor image of labeled N resolved by denaturing gel electrophoresis, and panel B represents the densitometric analysis of the bands using a Fuji2000 phosphor imager. Error bars represent standard deviations of two densitometric measurements from one experiment.

Fig. 5

One-step growth curve of IBV in Vero cells. A one-step growth curve was determined by determining the plaque forming units from 200 μl of 10-fold dilutions of IBV collected at varying times p.i. of Vero cells. The experiment was done in triplicate and the averages of three experiments are reported. Bars represent standard deviations.

Fig. 6

Graphic representation of the ³²P:³H cpm ratios of N protein obtained at varying hours p.i. from Vero- and CEK-infected cells and obtained from the purified virion from Vero and CEK cells at 16 h pi. The N was obtained by immunoprecipitation with rabbit anti-N polyclonal antibody, followed by TRIZOL treatment to remove bound RNA and by TCA precipitation to remove unincorporated label before determining the ³²P:³H cpm ratios. Standard deviations of two sets of experiments were less than 0.05. A significant linear trend (P < 0.0001) in the ratios was obtained.