Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [(35)S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.

FIG. 1.

Phosphorylation of the PRRSV N protein. (A) N protein phosphorylation in virus-infected cells. MARC-145 cells were infected with the PA8 strain (North American genotype) or Lelystad strain (European genotype) of PRRSV and labeled at 32 h p.i. for 6 h with either 50 μCi of [³⁵S]methionine/ml or 300 μCi of [³²P_i]orthophosphate/ml. For BCV, Mardin-Darby bovine kidney cells were infected and labeled at 24 h p.i. for 6 h with ³²P_i. Cell lysates were immunoprecipitated with a mixture of the N-specific MAbs for PA8 virus or with MAb NS99 for Lelystad virus. The BCV N protein was precipitated using MAb MD8-3. The precipitates were separated by SDS-12% PAGE followed by autoradiography. Lanes 1, 3, 5, and 7, uninfected; lanes 2 and 4, PA8 infected; lane 6, Lelystad virus infected; lane 8, BCV infected. (B) N protein phosphorylation in recombinant vaccinia virus-infected cells. MARC-145 cells were infected with either wild-type vaccinia virus (VV-WT) or recombinant vaccinia virus expressing the PRRSV N protein (VV-N) and radiolabeled at 12 h p.i. for 6 h with [³⁵S]methionine or ³²P_i. Cell lysates were immunoprecipitated as describe above. Lanes 9 and 11, wild-type vaccinia virus-infected cells; lanes 10 and 12, recombinant vaccinia virus-infected cells. (C) N protein phosphorylation in N gene-transfected cells. COS-1 cells were transfected with plasmids pCI-Neo or pCI-Neo-N containing the N coding sequence and labeled at 36 h posttransfection for 12 h with either [³⁵S]methionine or ³²P_i. Lanes 13 and 15, pCI-Neo transfected; lanes 14 and 16, pCI-Neo-N transfected. Arrows denote the N protein of BCV or PRRSV.

FIG. 2.

(A) Amino acid sequence and the predicted functional motifs of the PRRSV N protein. Nuclear localization signals are indicated in bold italics (27), and the protein kinase motifs for protein kinase C (PKC) (S-X-R) and PKA (R-X-S) are outlined with boxes. Numbers indicate the positions of serine residues. (B and C) Identification of phosphoamino acids of the N protein. MARC-145 cells infected with PRRSV (B) or recombinant vaccinia virus expressing the PRRSV N protein (C) were radiolabeled with 300 μCi of ³²P_i/ml and subjected to immunoprecipitation with an N-specific MAb mixture. The precipitates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. N protein bands were excised and hydrolyzed with 6N HCl for 2 h at 110°C. Amino acids were separated by one-dimensional electrophoresis on cellulose plates in 0.5% pyridine-5% acetic acid [pH 3.5] for 150 min at 400 V with cooling at 10°C. Unlabeled phosphoamino acid standards were visualized by staining with 0.25% ninhydrin (lanes 1 and 3), and the ³²P_i-labeled amino acids were visualized by autoradiography (lanes 2 and 4).

FIG. 3.

Subcellular distribution of the N protein. PRRSV-infected (25 h p.i.) (A) or N gene-transfected (B) cells were fixed with 4% formaldehyde and permeabilized with 0.1% NP-40. The cells were stained with an N-specific MAb mixture followed by Alexa Fluor 488 goat anti-mouse antibody and examined by laser scanning confocal microscopy. (C and D). Subcellular fractionation of the N protein. PRRSV (C) or recombinant vaccinia virus-infected (D) cells were radiolabeled with either [³⁵S]methionine (lanes 1 and 2) or ³²P_i (lanes 3 and 4) and separated into cytoplasmic (lanes 1 and 3) and nuclear (lanes 2 and 4) fractions followed by immunoprecipitation with an N-specific MAb mixture and SDS-PAGE.

FIG. 4.

Pulse-chase analysis of the PRRSV N protein. Following 1 h of starvation in deficient medium, PRRSV-infected cells were pulse-labeled at 32 h p.i. for 30 min in medium supplemented with either 150 μCi of [³⁵S]methionine/ml (A) or 500 μCi of ³²P_i/ml (B) and then chased for the indicated time intervals. At various time points postlabeling, cells were harvested and the lysates were reacted with an N-specific MAb mixture. The immunoprecipitated proteins were separated by SDS-12% PAGE and visualized by autoradiography. Mock-infected cells were harvested immediately following the pulse-label (lane 1). The chase times (in hours) are displayed above the panels.

FIG. 5.

Two-dimensional analysis and acid-urea gel electrophoresis of the PRRSV N protein. (A) For two-dimensional analysis, the N protein was purified by immunoprecipitation from virus-infected cells radiolabeled with 50 μCi of [³⁵S]methionine/ml. For the first dimension, the samples were applied to precast Immobiline dry strips (pH 6 to 11, linear pH gradient) and focused at 20°C for a total of 25,000 V-h. For the second-dimension separation, the equilibrated strips were placed on top of an SDS-15% polyacrylamide gel and electrophoresed at 15 mA for the first 15 min and 30 mA for a total of 5 h. The gel was dried and autoradiographed. (B) Analysis of the PRRSV N protein by acid-urea gel electrophoresis. MARC-145 cells were infected with PRRSV and radiolabeled with 50 μCi of [³⁵S]methionine/ml between 32 and 38 h p.i. Cell lysates were prepared and subjected to immunoprecipitation. The proteins were eluted from Sepharose beads in acid-urea sample buffer (5% acetic acid, 9 M urea, 0.002% methyl green) by heating samples at 80°C for 5 min. After prerunning gels in reverse polarity for 2 h at 150 V in 5% acetic acid, samples were loaded and electrophoresed at a constant voltage of 150 V for 1.5 h in a 15% polyacrylamide gel containing 6.4 M urea and 5% acetic acid. The gel was dried and autoradiographed. Lane 1, mock infected; lane 2, PRRSV infected.

FIG. 6.

Phosphorylation of the virion N protein. Virus-infected cells were labeled between 36 and 48 h p.i., and the culture supernatants were harvested. The supernatants were preclarified to remove cellular debris before concentrating the virus through a 20% sucrose cushion for 2 h at 25,000 rpm in an SW28 rotor (XL-90 ultracentrifuge; Beckman Instruments, Inc.). Virus pellets obtained from the 20% sucrose cushion were resuspended in Tris-EDTA buffer and further purified in a 20-to-50% continuous sucrose gradient at 50,000 rpm (SW55Ti) for 5 h. Fractions were taken from the top of the gradients and aliquots from alternate fractions were directly analyzed without immunoprecipitation (A and B) or immunoprecipitated using N-specific MAbs (C), followed by SDS-PAGE under reducing conditions and autoradiography. (A) [³⁵S]methionine-labeled virions; (B and C) ³²P_i-labeled virions. GP5[³⁵S], [³⁵S]methionine-labeled GP5 glycoprotein; M[³⁵S], [³⁵S]methionine-labeled membrane-associated protein; N[³⁵S], [³⁵S]methionine-labeled nucleocapsid protein; N[³²P], [³²P]phosphate-labeled nucleocapsid protein.