Expression of viral protein and RNA from cells infected with rPIV5-CPI− or rPIV5-CPI+. (A and B) Determining expression of viral protein from cells infected with rPIV5-CPI− or rPIV5-CPI+ using flow cytometry. BSR T7 or HeLa cells were infected with mock, wt PIV5, rPIV5-CPI−, or rPIV5-CPI+ at a MOI of 3. The cells were collected, fixed, and stained with anti-HN antibody at 16 hpi. A flow cytometer was used to gate HN-positive cells, and then the mean fluorescence intensities for HN were measured and graphed. Error bars are standard deviations of means. Panel A shows HeLa cells; panel B shows BSRT7 cells. (C and D) Determining expression of viral protein from cells infected with rPIV5-CPI− or rPIV5-CPI+ using immunoprecipitation. The infected cells were labeled and immunoprecipitated with anti-PIV5 antibodies as described in Materials and Methods. Panel C shows HeLa cells; panel D shows BSR T7 cells. (E to G) Viral RNA synthesis in cells infected with rPIV5-CPI− or rPIV5-CPI+. HeLa cells were infected with wt PIV5, rPIV5-CPI−, or rPIV5-CPI+. At the indicated hours postinfection, cells were lysed and total RNA extracted. Real-time RT-PCR was performed to determine the relative levels of the mRNA or viral RNA. Panels E and F show relative viral RNA and mRNA levels of cells infected with PIV5, rPIV5-CPI−, or rPIV5-CPI+ at different time points, obtained by comparing the RNA level at each time point with that of the 0-hpi sample of the corresponding virus. (G) Ratios of mRNA to viral RNA levels. Error bars are the standard deviations of means.

Elevated minigenome replication with Pcpi− and Pcpi+. A minigenome plasmid (pSMG-RL) that contains a Renilla luciferase reporter gene described previously (23) was modified to comply with the rule of six as described in the Materials and Methods. A negative-sense minigenome was generated from T7 RNA polymerase transcription in BSR T7 cells. In the presence of NP and L and with type P (Pwt), P from rPIV5-CPI+ (Pcpi+) (A) or P from rPIV5-CPI− (Pcpi−) (C), this negative-sense RNA template is replicated and transcribed to give rise to the reporter gene mRNA, resulting in luciferase activity. The pT7-F-Luc plasmid, which contains an F-Luc reporter gene as a transfection efficiency control, was transfected along with the plasmids. Firefly and Renilla luciferase activities were detected in cell lysates at 18 to 20 h posttransfection, as described in Materials and Methods. The relative luciferase activities are calculated at ratios of Renilla luciferase activity (indicative of minigenome replication) versus F-Luc activity (indicative of transfection efficiency). Due to the quality and amount of plasmids used in the experiments and passages of BSR T7 cells, which affect expression of T7 RNA polymerase, the relative activity fluctuates. To ensure the validity of comparisons among different P proteins, we have used a spectrum of concentrations of the P proteins in the experiments. Cell lysate aliquots from panel A or B were subjected to immunoblotting using anti-NP or anti-P antibody, respectively (as described in Materials and Methods). NC, negative control, containing all plasmids except L encoding plasmid. Error bars are the standard deviations of means from examples containing six replicates for each transfection. (C) Inhibition of minigenome replication by Vcpi−. Increasing amounts of V or Vcpi− expression plasmid were cotransfected with equal amounts of P or Pcpi− expression plasmid in the PIV5 minigenome system as described above. The total amount of DNA transfected was kept constant by using a green fluorescent protein expression plasmid. Cell lysate aliquots were collected and subjected to immunoblotting using anti-NP and anti-P antibodies. NC, negative control, lacking L. Error bars are the standard deviations of means from examples containing six replicates for each transfection.

Transcription activities of P, Pcpi−, and Pcpi+. (A) A replication-deficient minigenome plasmid, pMG-m-R-Luc (23), was transfected into BSR T7 cells together with increasing amounts of plasmid encoding P, Pcpi−, or Pcpi+, along with a plasmid encoding NP and L. Firefly and Renilla luciferase activities were measured in cell lysates 18 to 20 h posttransfection. (B) Cell lysate aliquots were collected and subjected to immunoblotting using anti-NP or anti-P antibody. NC, negative control without L. Error bars are the standard deviations of means from examples containing six replicates for each transfection.

Activities of mutant P proteins in minigenome system. (A) Increasing amounts of plasmids encoding P and different P mutant proteins that contain double or single substitution mutations at amino acid positions 32, 33, and 157 (Table 1) were transfected along with other plasmids of the PIV5 minigenome system. Replication of the minigenome systems was measured as described in Materials and Methods. (B) Cell lysate aliquots from P and S157F protein-expressing cells were collected and subjected to immunoblotting using anti-NP or anti-P antibody. NC, negative control which lacks L. Error bars are standard deviations of means from examples containing six replicates for each transfection.

Interactions between P and P mutant with NP or L and oligomer formation of P. (A and B) Coimmunoprecipitation of P and P mutants with either L (with two Flag tags) or NP, respectively. BSR T7 cells were transfected with empty plasmid or plasmids encoding P, Pcpi−, Pcpi+, S157F, and/or L (A) or and/or NP (B). At 24 h posttransfection, the cells were metabolically labeled with ³⁵S-ProMix, lysed, and then subjected to immunoprecipitation with anti-P and anti-NP antibody (A) or anti-Flag antibody for L (B). The precipitates were then resolved in 10% SDS-PAGE and visualized by using a Storm PhosphorImager. (C) P protein oligomer formation. P and P mutants were expressed in BSR T7 cells, metabolically labeled, and then cross-linked using disuccinimidyl tartrate or dimethylsulfoxide as a control. The cells were lysed then subjected to immunoprecipitation with anti-P antibody. The precipitates were mixed with protein lysis buffer in the presence or absence of DTT, resolved in 10% SDS-PAGE, and visualized as described in Materials and Methods.

Phosphorylation of P in cells infected with rPIV5-CPI−, rPIV5-CPI+, or PIV5. BSR T7 cells were mock infected or infected with wt PIV5, rPIV5-CPI−, or rPIV5-CPI+ at a MOI of 3. At 18 to 20 hpi, the infected cells were labeled with ³⁵S-ProMix or [³³P]orthophosphate for 4 h. The cells were lysed and immunoprecipitated with anti-P antibody. The P proteins were immunoprecipitated and resolved in 10% SDS-PAGE (A). The results of three experiments were averaged and graphed (B). The level of wt P phosphorylation, defined as the intensity of ³³P/³⁵S, is set as 1. Error bars are standard deviations of means. The average reduction in phosphorylation of the P protein from three experiments is graphed (P = 0.02).

Determination of phosphorylation sites within the P protein. HeLa cells were infected with wt PIV5 at a MOI of 3. The P protein was immunoprecipitated from cell lysates using anti-V5-conjugated agarose (Sigma-Aldrich) which recognizes the P protein and resolved by 10% SDS-PAGE as described in Materials and Methods. The band corresponding to the P protein was excised, digested with trypsin, and then processed to enrich phosphopeptides using TiO₂. Both the enriched fraction and the flowthrough (which contains all of the nonphosphorylated peptides) were analyzed by LC-MS/MS on a Waters Q-Tof Ultima mass spectrometer. The graph represents the LC-MS/MS product ion spectrum of the parent ion of phosphorylated peptide 144-163 from the trypsin digest of the PIV5 P protein. The y_n-98 ion corresponds to the natural loss of H₃PO₄ from the parent ion. The b- and y-type fragment ions observed are shown with the peptide sequence (insert box). The x axis and y axis show mass-to-charge ratio (m/z) and relative abundance of the ions (% relative intensity), respectively.

Activities of S157A and S157D proteins. Increasing amounts of plasmids encoding P (wt) or a P mutant (S157A or S157D) were transfected along with other plasmids of the PIV5 minigenome system. Replication of the minigenome systems was measured as described in Materials and Methods. Neg, negative control without L. Error bars are standard deviations of means from examples containing six replicates for each transfection.