Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection

Expression of M2-1 protein phosphorylation mutants. (A) Analysis of mutant proteins. HEp-2 cells, infected with vTF7-3, were either mock transfected or transfected with 5 μg of plasmids expressing M2-1 or the indicated phosphorylation mutants (S58A, S61A, T56A, or T56A S58A). The cells were labeled with either [³⁵S]methionine and [³⁵S]cysteine (³⁵S) or inorganic [³³P]phosphate (³³P) for 3 h at 17 h p.t. Cytoplasmic extracts were prepared and precleared. Then proteins were immunoprecipitated with MAb ICI3, separated by SDS-PAGE, and subjected to autoradiography. After immunoprecipitation, the samples in lanes 13 and 14 were treated with 200 U of λ-PPase for 30 min at 30°C. The bracket indicates the position of the ³³P-labeled coimmunoprecipitated material. (B) RS virus proteins. RS virus-infected (RS inf.) HEp-2 cells, harvested 24 h post infection, were prepared and immunoprecipitated as described for panel A, except the lysate was not precleared. (C) In vitro CKI phosphorylation of M2-1 protein. M2-1 protein produced in E. coli was incubated with CKI and [γ-³³P]ATP for 30 min at 37°C. The protein was separated by SDS-PAGE on an 11% polyacrylamide gel and Coomassie stained (left) or subjected to autoradiography (right).

Effect of M2-1 protein phosphorylation mutations on transcription. (A) Diagram of F/M2 replicon containing the authentic F/M2 gene junction and the expected products of transcription. (B) MVA-T7-infected HEp-2 cells were transfected with pF/M2, pN, pP, pL, and increasing amounts of M2-1 wt or mutant plasmids (0.15 to 0.6 μg), pT56A, pS58A, pS61A, or pT56AS58A. The cells were labeled for 5.5 h at 17 h p.t. with [³H]uridine in the presence of Act D. The purified RNAs were visualized by separation on an agarose-urea gel followed by fluorography. (C) Quantitation of percent readthrough products at each amount of input plasmid. Percent readthrough was determined by densitometric analysis and conversion to molar amounts of RNA based upon [³H]uridine incorporation. Rep, replication; le, leader; tr, trailer; ig, intergenic; AAAn, polyadenylate.

RNA is coimmunoprecipitated with the M2-1 protein. (A) HEp-2 cells infected with vTF7-3 were either mock transfected or transfected with 5 μg of pM2-ORF1 or plasmids expressing mutant M2-1 proteins. The cells were labeled for 3 h at 17 h p.t. with inorganic [³³P]phosphate. Immunoprecipitations were performed using MAb ICI3. The immunoprecipitated proteins were analyzed by SDS-PAGE on an 11% polyacrylamide gel followed by autoradiography. (B) Digestion of ³³P-labeled component. Transfected (lane 3, mock; lanes 4 to 7, 5 μg of pM2-ORF1) and infected (lanes 1 and 2) cells were prepared as for panel A, except 5 μg of proteinase K (Prot K), 5 μg of RNase A, or 1 U of DNase RQI was added after immunoprecipitation as indicated, followed by incubation for 30 min at 37°C.

Analysis of coimmunoprecipitated RNA from an RSV infection. HEp-2 cells in 100-mm-diameter dishes were infected with RS virus (MOI = 10). The cells were labeled in the presence of Act D at 17 h postinfection for 5 h with inorganic [³³P]phosphate. Cytoplasmic extracts were prepared, and lysates were either immunoprecipitated with MAbs to the M2-1, N, or F protein or analyzed for total RNA. RNAs were released from the immunoprecipitation mixture by boiling it in NTE. Immunoprecipitated and total RS virus RNAs were purified by phenol extraction and ethanol precipitation, followed by separation on an agarose-urea gel and fluorography. The locations of the various RS virus monocistronic and polycistronic RNAs are indicated on the left. +, present; −, absent.