JOURNAL OF VIROLOGY, Sept. 2008, p. 9123–9133
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 18
A Single Amino Acid Residue Change in the P Protein of
Parainfluenza Virus 5 Elevates Viral Gene Expression?
Khalid A. Timani,1† Dengyun Sun,2Minghao Sun,3‡ Celia Keim,1§ Yuan Lin,1Phuong Tieu Schmitt,1
Anthony P. Schmitt,1,2,3,4and Biao He1,2,3,4*
Department of Veterinary and Biomedical Sciences,1Intercollege Graduate Program in Cell and Developmental Biology,2
Graduate Program in Pathobiology,3and Center of Molecular Immunology and Infectious Disease,4
Pennsylvania State University, University Park, Pennsylvania 16802
Received 8 February 2008/Accepted 3 July 2008
Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA
species through a process of pseudotemplated insertion of two G residues at a specific site during transcription,
resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously
it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5
(rPIV5-CPI?) that exhibits elevated viral protein expression and induces production of cytokines, such as beta
interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and
the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI? are due to mutations in the
P protein and/or mutations in the V protein. To address this question, we used a minigenome system and
recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that
the P protein with six amino acid residue changes (Pcpi?) was more efficient than wild-type P in facilitating
replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi?) still inhibits
minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene
expression in rPIV5-CPI? virus-infected cells can be attributed to a P protein with an increased ability to
facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157
of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi?) is sufficient for
elevated viral gene expression. Using mass spectrometry and33P labeling, we found that residue S157 of the
P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at
residue 157 plays an important role in regulating viral RNA replication.
Parainfluenza virus 5 (PIV5), formerly known as simian virus
5, is a prototypical paramyxovirus in the Rubulavirus genus of
Paramyxovirinae (4). The paramyxovirus family includes many
important human and animal pathogens, such as Sendai virus,
mumps virus, human parainfluenza viruses 1, 2, 3, and 4, New-
castle disease virus, measles virus, and emerging viruses, such as
Hendra virus and Nipah virus (19). Paramyxoviruses contain non-
segmented negative-stranded RNA genomes, which are encap-
sidated by nucleocapsid protein (NP). The gene order within the
paramyxovirus genomes is 3?-NP-P(V/W/C)-M-F-(SH)-HN-L-5?,
where genes in parenthesis are not found in all species (reviewed
in reference 19). The viral RNA-dependent RNA polymerase of
paramyxoviruses minimally consists of two proteins, phosphopro-
of paramyxoviruses have masses of 220 to 250 kDa. They have the
capacity to initiate, elongate, and terminate transcription. In ad-
dition, they have the capacity to insert nontemplated G residues
at selected sites within viral mRNAs during transcription and to
add cap structures to the 5? ends of viral transcripts. While the L
protein of the P-L complex of viral RNA-dependent RNA poly-
merase is thought to contain polymerase activity, the P protein is
thought to be a regulatory protein without intrinsic enzymatic
activity. The P protein of paramyxoviruses is phosphorylated
(hence the name phosphoprotein). The phosphorylation of P in
nonsegmented negative-stranded RNA virus is thought to play a
critical role in virus RNA synthesis (19). Mutations within P of
vesicular stomatitis virus that selectively affect viral RNA replica-
tion or viral mRNA transcription have been identified, suggesting
that the protein plays an important role in regulating viral RNA
replication and viral mRNA transcription (7). However, the roles
of phosphorylation of paramyxovirus P proteins in viral RNA
genome systems indicate that mutating putative phosphorylation
when these mutations are incorporated into virus genomes, no
effect on virus RNA synthesis has been observed, leading to the
theory that phosphorylation of the P protein does not have a role
in viral RNA synthesis (17, 18, 26). Recently studies using recom-
binant PIV5 expressing an additional copy of the P gene suggest
antiviral responses (9, 13). However, it is not clear whether the
putative role of the PIV5 P protein in preventing host innate
* Corresponding author. Mailing address: Center of Molecular Im-
munology and Infectious Disease, Department of Veterinary and Bio-
medical Sciences, Pennsylvania State University, 115 Henning Bldg.,
University Park, PA 16802. Phone: (814) 863-8533. Fax: (814) 863-
6140. E-mail: email@example.com.
† Present address: Indiana University School of Medicine, Depart-
ment of Microbiology and Immunology, R2 302, 950 W. Walnut St.,
Indianapolis, IN 46202.
‡ Present address: Division of Oncovirology, Department of Molec-
ular and Experimental Medicine, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037.
§ Present address: Department of Pathology & Cell Biology, Colum-
bia University, 630 W. 168th Street, New York, NY 10032.
?Published ahead of print on 9 July 2008.
immune responses is direct or indirect due to the P protein’s role
in viral RNA synthesis.
The V/P gene of PIV5 can be transcribed into two species of
mRNA in about equal amounts through a process of pseudo-
templated nucleotide insertion, in which two G residues are
inserted at a specific location during viral RNA transcription.
The faithful transcription of the gene results in a population of
mRNAs encoding the V protein, whereas insertion of the non-
templated two G residues results in a population of mRNAs
encoding the P protein. The V protein (222 amino acid resi-
dues) and the P protein (392 amino acid residues) have iden-
tical N termini (164 amino acid residues) (39). The V protein
is a structural component of PIV5 virions (?350 molecules per
virion) and is multifunctional (30). The V protein C-terminal
domain contains seven cysteine residues, resembling a zinc
finger domain, and binds atomic zinc (25, 30, 36, 39). It inter-
acts with soluble NP (32), and the N-terminal domain binds
RNA through a basic region (21). The PIV5 V protein inter-
acts with a cellular protein (DDB1). This interaction requires
the presence of the C-terminal domain of the V protein (20).
Expression of the PIV5 V protein slows down the cell cycle in
a manner that is dependent on the C-terminal region of the
protein (20). Coexpression of DDB1 can partially restore the
changes in the cell cycle caused by V (20). The V protein of
PIV5 can cause degradation of the STAT1 protein, an essential
regulator of interferon (IFN) signaling, through a proteasome-
mediated pathway in human cells but not in mouse cells (8). It
has been shown that V, DDB1, Cul4A, STAT1, and STAT2
form a complex, which is essential for V-mediated STAT1
degradation, and the V protein has an E3 ubiquitin ligase
activity (28, 40). In addition to preventing IFN signaling, the V
protein can inhibit beta IFN (IFN-?) production through an
IRF-3-dependent pathway (15, 31) and block interleukin 6
(IL-6) expression in virus-infected cells (24). Recently it was
shown that the V protein interacts with MDA-5 and that this
interaction plays an important role in blocking activation of
IFN-? production (1). The V protein is also known to play an
essential role in blocking apoptosis in virus-infected cells.
While the exact mechanism is not clear, it is thought that the V
protein blocks endoplasmic reticulum stress-induced apoptosis
and this blockage function is independent of its involvement
with IFN pathways (38). Using a minigenome system devel-
oped for PIV5 free of vaccinia virus infection, it has been
reported that the expression of the V protein inhibits mini-
genome replication by inhibiting viral RNA synthesis (23).
Further investigation of the mechanism indicated that the V
protein interacts with Akt1, a serine/threonine kinase, and the
V protein likely exerts its influence on viral RNA synthesis via
its interaction with Akt1 (37).
A strain of PIV5 causing a neurological disorder in canines
was isolated by Evermann et al. (12) and is called canine
parainfluenza virus (CPI?). During the course of studying the
CPI? virus, a derivative of CPI?, termed CPI?, was isolated
from a dog that was experimentally infected with the CPI?
virus (2). It was found that there are differences among the V/P
genes of CPI?, CPI?, and the commonly used lab strain W3A,
in addition to differences in other viral genes (4, 5, 35). The
CPI? virus has eight amino acid residues in the V/P gene that
are different from those of the W3A strain (referred to as the
wild type [wt] in this work), and six of them are in the shared
region of the V and P proteins. The CPI? virus has five amino
acid residues that are different from those of wt PIV5 in the
V/P gene, and three of them are in the shared region of the V
and P proteins. Wansley et al. generated recombinant PIV5
based on the W3A strain but containing six amino acid residue
changes in the shared region of the V and P proteins, corre-
sponding to the CPI? virus. This virus was termed rPIV5-
CPI? (42). They observed that rPIV5-CPI? causes elevated
viral gene expression, induces expression of host antiviral re-
sponse genes, such as IFN-? and IL-6, and induces apoptosis
of infected cells compared to the wt W3A strain. Because the
six mutations are within the shared region of the V and P
proteins, it is not clear whether the phenotypes associated with
rPIV5-CPI? are due to mutations in the P protein or muta-
tions in the V protein. To address this question, we used both
a vaccinia virus-free minigenome system that we developed
and recombinant viruses to study the effects of mutations on
the functions of the P and V proteins.
MATERIALS AND METHODS
Plasmids, viruses, and cells. Plasmids used in this work were constructed using
standard molecular cloning techniques. Details of the construction of the plas-
mids and the computer sequence files of the plasmids are available on request.
The plasmid pSMG-RL, which contains a PIV5 minigenome system that com-
plies with the rule of six, was constructed from pMG-RL (23) by adding three
nucleotides between the reporter gene and the leader sequence. Mutant P genes
encoding V32I, T33I, S157F, S157A, S157D, V32I-T33I, T33I-S157F, or V32I
S157F were made from a copy of P from the W3A strain (23) (Table 1) by using
four-primer PCR as described previously (14). The P mutants were cloned into
the pCAGGS expression vector (27). An L protein with two copies of Flag
epitope tags at its C terminus was constructed in the pCAGGS background for
expression and detection in coimmunoprecipitation experiments. Plasmids con-
taining full-length viral genomes corresponding to rPIV5-CPI? and rPIV5-
CPI? were generated by introducing six or three substitution mutations, located
at amino acid residue positions 26, 32, 33, 50, 102, and 157 or 32, 33, and 157,
respectively, in the V/P sharing region of the rPIV5 genome (Table 1) in a
plasmid containing the PIV5 genome, pBH276, which contains the wt W3A
full-length genome, as previously described (16). The viruses (rPIV5-CPI? and
rPIV5-CPI?) were rescued following a procedure described by He et al. and
Waning et al. (16, 41). The recovered viruses were plaque purified, and the V/P
gene sequence was confirmed by reverse transcription (RT)-PCR sequencing.
The viruses were grown in Vero cells and titrated in BHK cells as previously
HeLa and Vero cells were grown in Dulbecco modified Eagle medium
(DMEM) (Gibco-BRL) containing 10% fetal calf serum. BHK cells were grown
TABLE 1. Amino acid residue substitutions encoded in
V/P gene of PIV5a
Amino acid residue at position:
26 32 3350 102157
aBoldfacing indicates residues associated with CPI?.
bEffectiveness indicates the ability of the P protein to facilitate viral RNA
9124TIMANI ET AL. J. VIROL.
in DMEM containing 10% fetal calf serum and 10% tryptose phosphatase broth.
BSR T7 cells were growth in the same medium as BHK cells, with the addition
of 400 ?g/ml G418 to maintain the expression of T7 RNA polymerase (3). All
cell lines were maintained in 100 IU/ml penicillin–100 ?g/ml streptomycin and
incubated at 37°C in 5% CO2.
Flow cytometry. BSR T7 or HeLa cells were mock infected or rPIV5, rPIV5-
CPI?, or rPIV5-CPI? infected at a multiplicity of infection (MOI) of 3. The
cells were collected at 16 h postinfection (hpi) and fixed with 0.5% formaldyhyde
for 2 h. The fixed cells were pelleted by centrifugation and then resuspended in
500 ?l of solution of fetal bovine serum (FBS)-DMEM (50:50). The cells were
permeabilized in 70% ethanol overnight. The cells were washed once with phos-
phate buffered saline deficient in Mg2?and Ca2?(PBS?) and then incubated
with mouse monoclonal anti-hemagglutinin-neuraminidase (HN) antibody in
PBS? containing 10% FBS for 30 min at room temperature. The cells were
stained with antimouse antibody labeled with phycoerythrin for 30 min at room
temperature in the dark and then washed once with PBS? containing 10% FBS.
The fluorescence intensity was measured using a flow cytometer.
Transfection and dual luciferase assay. To measure the activities of the P
proteins, a modified minigenome system of PIV5 was used. The BSR T7 cells
were seeded in 24-well culture plates. The cells were transfected at 70 to 80%
confluence with a total of 0.827 ?g of plasmid DNA per well containing
pCAGGS-NP, pCAGGS-L, pSMG-R-luc (minigenome plasmid in which the
number of nucleotides from the trailer to leader sequence is a multiple of six),
pCAGGS-P or -P mutants and/or p-CAGGS-V or pCAGGS-Vcpi? (which has
six substitution mutations in the V/P shared region [Table 1]). The total amounts
of transfected plasmids were kept constant using pCAGGS-GFP. Plasmids were
transfected using Lipofectamine Plus (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instructions. To measure the transfection efficiency in the
luciferase assay, an equal amount of the pT7-F-Luc plasmid, which contains a
firefly luciferase (F-Luc) reporter gene, was included in the DNA transfection
mixture. At 18 to 20 h posttransfection, the cells were lysed in 140 ?l passive lysis
buffer (Promega). Twenty microliters of lysate from each well was then used in
the subsequent dual luciferase assay, according to the manufacturer’s protocol
(Promega). To examine the transcription activity of P and P mutants, a mini-
genome with a defective trailer sequence that is active only in transcription was
used as described by Lin et al. (23). Minigenome replication and/or transcription
was normalized for each sample using dual luciferase activity. The relative
luciferase activity is defined as the ratio of R-Luc to F-Luc activities.
Western blot. An aliquot of the cell lysate from the dual luciferase assay was
mixed with an equal volume of 2? protein lysis buffer (60 mM Tris-HCl [pH 6.8],
40% glycerol, 4% sodium dodecyl sulfate [SDS], 3% dithiothreitol [DTT], and a
few grains of bromophenol blue) as previously described (29). Samples were
resolved in 10 or 15% SDS-polyacrylamide gel electrophoresis (PAGE) and
transferred onto a polyvinylidene difluoride membrane. Immunoblotting using
antibodies against PIV5 proteins was performed as previously described (22, 38).
Immunoprecipitation. To examine the expression levels of viral proteins in
infected cells, cells (HeLa or BSR T7) were mock infected or infected with PIV5,
rPIV5-CPI?, or rPIV5-CPI? at a MOI of 3 as before. At 16 hpi, the infected
cells were starved in DMEM lacking cysteine-methionine for 30 min and then
labeled with35S-Promix (Amersham Life Sciences) (10 ?Ci/ml) for 3 h. The cells
were lysed with whole-cell extract buffer (WCEB) (50 mM Tris-HCl [pH 8], 280
mM NaCl, 0.5% NP-40, 0.2 mM EDTA, 2 mM EGTA, and 10% glycerol) (40),
and the aliquots were coimmunoprecipitated using antibodies against PIV5 NP,
P, and M. The precipitated proteins were resolved by 10% SDS-PAGE and
visualized using a Storm PhosphorImager (Molecular Dynamics Inc., Sunnyvale,
CA). To study the interaction of P and P mutants with either NP or L, plasmids
encoding P or P mutants with NP or with L containing two copies of Flag epitope
tags at the C terminus encoded by the L gene to facilitate its detection were
cotransfected into cells. At 20 to 24 h posttransfection, the cells were labeled and
immunoprecipitated using anti-P, anti-NP (NP-214) (33), or anti-Flag M2
Chemical cross-linking. The P and P mutants were expressed in BSR T7 cells
and then labeled with35S-Promix as before. Chemical cross-linking was per-
formed as described previously (34). Briefly, metabolically labeled cells were
washed with PBS? and removed from the dish with PBS? containing 50 ?M
EDTA. Cells were pelleted by centrifugation and resuspended in PBS?. Cross-
linking reactions were carried out in 200-?l aliquots of cell suspension in 0.5%
NP-40. One hundred millimolar disuccinimidyl tartrate (Pierce, Rockford, IL)
stock solution was dissolved in dimethylsulfoxide and added to the cross-linking
reaction mixture so that the final concentration was 1 mM. Cross-linking reac-
tions were incubated for 2 h at 4°C. The cells were lysed, and the proteins
precipitations were processed as mentioned earlier. Samples were mixed with an
equal volume of 2? protein lysis buffer in the presence or absence of DTT and
then resolved by 10% SDS-PAGE.
Phosphorylation of P. To investigate the phosphorylation level of P and P
mutants in the infected cells, BSR T7 cells were mock infected or rPIV5, rPIV5-
CPI? and rPIV5 CPI? infected at a MOI of 2. At 18 to 20 hpi, the cells were
starved with either DMEM lacking cysteine-methionine or DMEM lacking phos-
phate and then labeled with 100 ?Ci35S-Promix or 200 ?Ci [33P]orthophosphate
(PerkinElmer), respectively, for 4 h. The immunoprecipitations using anti-P
antibody (33) were processed as described above.
Real-time PCR. Monolayer cells in 6-cm dishes were washed with PBS and
inoculated with PIV5, rPIV5-CPI?, or rPIV5-CPI? viruses in DMEM with 1%
bovine serum albumin at a MOI of 5 for 1 to 2 h at 37°C. Cells were then washed
and incubated in DMEM with 2% FBS at 37°C with 5% CO2. At 0, 4, 8, 12, 16,
and 20 hpi, total RNAs were extracted from infected cells using an RNeasy
minikit (Qiagen) and eluted in 100 ml RNase-free H2O. Five milliliters of total
RNA for each sample was used for the RT reaction with Superscript III reverse
transcriptase (Invitrogen) according to the manufacturer’s protocol. Oligo(dT)15
was used to measure mRNA levels; oligonucleotide BH191, which hybridizes to
the viral RNA of PIV5 within the HN gene, was added for detection of viral
RNA levels. Five percent of the cDNA from each sample was then used for every
real-time PCR on an ABI 7300 real-time PCR system using Taqman Universal
PCR master mix (Applied Biosystems) and custom-made Taqman gene expres-
sion assays (Applied Biosystems) for the HN gene. Primers were as follows:
forward primer, GGGTACTAGATGTATGGGCAACA; reverse primer, ACG
CCGCCATATATTGGAAAGAG; reporter, CCCCGCTTCCTGTTCC, with 6-
carboxyfluorescein dye and NFQ quencher. Results were analyzed with the RQ
study software program (Applied Biosystems) to obtain threshold cycle values.
Relative levels of mRNA and viral RNA at each time point were determined by
calculating the change in the threshold cycle, with 0-hpi samples infected with the
same virus serving as the calibrators. The ratios of mRNA and viral RNA levels
of the same samples were calculated by setting the viral RNA levels as calibrat-
ors. Each sample was done in quadruplicate.
Mass spectroscopic analysis. To determine the phosphorylated residues in the
PIV5 P protein, mass spectroscopic analysis was performed. HeLa cells in 10-cm
plates were mock or PIV5 infected at a MOI of 3. At 24 hpi, the cells were lysed
with WCEB buffer and the P protein was immunoprecipitated by using anti-V5
agarose-conjugated beads (Sigma-Aldrich) for 4 h at 4°C or overnight. The beads
were washed three times with WCEB buffer, mixed with 2? protein lysis buffer
in the absence of DTT, and then resolved by 10% SDS-PAGE. The band
corresponding to the P protein was excised and digested with trypsin (12.5 ng/?l
in 25 mM NH4HCO3) at 37°C for 16 h. The digested peptides were further
processed for phosphopeptide enrichment using TiO2.Both the enriched frac-
tion and the flowthrough (which contains all of the nonphosphorylated peptides)
were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/
MS) on a Waters Q-Tof Ultima mass spectrometer at Yale Cancer Center Mass
Spectrometry Resource and W.M. Keck Foundation Biotechnology Resource
Laboratory, Yale University. All MS/MS spectra were searched using the auto-
mated Mascot algorithm against the NCBI database for possible tyrosine (Tyr),
threonine (Thr), and serine (Ser) phosphorylation sites.
Elevated viral protein expression and viral genome replica-
tion by rPIV5-CPI? and rPIV5-CPI?. It has been shown that
six amino acid residue substitutions within the V/P gene are
sufficient to cause elevated viral gene expression (42). To in-
vestigate the roles of individual residues in increasing viral
gene expression, we have generated a recombinant PIV5 en-
coding the P protein that has the same three amino acid res-
idue substitutions, relative to the wt P protein sequence, that
are found in CPI? virus (4). We call it rPIV5-CPI? (Table 1).
HeLa cells, a human cell line, and BSR T7 cells, a murine cell
line, were infected with PIV5, rPIV5-CPI?, or rPIV5-CPI?.
To examine expression of viral proteins in individual cells, HN
protein expression was quantified using flow cytometry (Fig.
1A and B). As expected, rPIV5-CPI? caused increased expres-
sion of viral protein compared with wt PIV5 infection, consis-
tent with the previous report (42). Interestingly, infection with
rPIV5-CPI? also caused increased expression of viral protein
VOL. 82, 2008 ROLE OF PIV5 P PROTEIN IN VIRAL REPLICATION9125
FIG. 1. ExpressionofviralproteinandRNAfromcellsinfectedwithrPIV5-CPI?orrPIV5-CPI?.(AandB)Determiningexpressionofviralprotein
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.
compared with wt PIV5 infection, indicating that three amino
acid residue substitutions (V32I, T33I, and S157F) within the
shared region of the V and P proteins are sufficient to convert
wt PIV5 into a virus that expresses its proteins at higher levels.
It is known that wt PIV5 causes degradation of STAT1 in
human cells, such as HeLa cells, but not in BHK cells. Since
the same results have been obtained in both human HeLa cells
and BSR T7 cells, a derivative of BHK cells, it is unlikely that
the ability of virus to cause degradation of STAT1 plays a role
in the increased expression of viral proteins by rPIV5-CPI?
and rPIV5-CPI?. To further confirm the results, immunopre-
cipitation of viral proteins from infected HeLa or BSR T7 cells
was performed. Consistent with flow cytometry data, both
rPIV5-CPI? and rPIV5-CPI? virus infections resulted in a
higher level of viral gene expression (Fig. 1C and D). These
results also indicate that the increased viral gene expression is
not cell type specific.
The increased viral gene expression could potentially be
attributed to increased viral RNA replication, since this would
increase the amount of viral RNA genome for use as a tem-
plate during transcription. Another possibility is that increased
viral gene expression results from increased RNA transcrip-
tion. To investigate the mechanism for the increased viral gene
expression in rPIV5-CPI? and rPIV5-CPI?, we examined vi-
ral RNA synthesis in infected cells. HeLa cells were infected
with mock, PIV5, rPIV5-CPI?, or rPIV5-CPI? at a MOI of 3.
At different time points after infection, RNAs were purified
from infected cells and measured using a real-time RT-PCR
assay. Both rPIV5-CPI? and rPIV5-CPI? infection resulted
in much higher levels of viral RNA genome production, indi-
cating that viral RNA replication increased in cells infected
with rPIV5-CPI? or rPIV5-CPI? compared with that in
PIV5-infected cells (Fig. 1E), consistent with the observation
that higher viral protein expression levels were observed in
cells infected with rPIV5-CPI? or rPlV5-CPI? than in wt
virus-infected cells. While overall levels of viral transcripts are
higher in cells infected with rPIV5-CPI? or rPIV5-CPI? than
in PIV5-infected cells, as expected (Fig. 1F), transcription of
viral mRNA normalized to the amount of viral genome RNA
remains similar among all virus-infected cells, indicating that
the increase in activities of Pcpi? and Pcpi? proteins results
from increased RNA genome replication (Fig. 1G).
Elevated minigenome replication in the presence of Pcpi?
and Pcpi?. Previously we generated a minigenome system free
of vaccinia virus to study viral RNA synthesis of PIV5 (23). To
further study the mechanism of increased viral gene expression
in cells infected with rPIV5-CPI? or rPIV5-CPI?, we com-
pared the activity of the P protein of rPIV5-CPI? (Pcpi?) and
the P protein of rPIV5-CPI? (Pcpi?) with the P protein of the
wt (P or Pwt) in the minigenome system. Because overexpres-
sion of the P protein inhibits minigenome replication, an ob-
servation that has been reported previously for PIV5 and other
systems (23), we used a spectrum of P-protein concentrations
to ensure that differences in minigenome expression reflect
differences in P-protein activities, not differences in P-protein
quantity. As expected, initially, increased expression of the P
protein resulted in increased reporter gene expression, and
further overexpression of the P protein inhibited minigenome
replication (Fig. 2A). Using the same spectrum of protein
concentrations, Pcpi? gave rise to higher minigenome re-
porter expression than wt P, indicating that Pcpi? is more
efficient in this system than the wt P protein. Further overex-
pression of Pcpi? inhibited minigenome replication, similar to
the effect observed with the wt P protein. All P protein expres-
sion levels in minigenome replication experiments were mea-
sured by immunoblotting (Fig. 2A, bottom panel). Using the
same approach, Pcpi? was found to be more efficient for
minigenome replication than the wt P protein. The results
suggest that a more efficient P protein in rPIV5-CPI? or
rPIV5-CPI? contributes to the increased viral protein expres-
sion in cells infected with rPIV5-CPI? or rPIV5-CPI?. This
result obtained from the minigenome system is consistent with
observations made for virus-infected cells. Thus, three amino
acid residues within the identical N-terminal region of the V
and P proteins contribute to the increased activity of the P
protein of rPIV5-CPI?.
Vcpi? retains its ability to inhibit minigenome replication.
Previous work from our laboratory demonstrated that the V
protein of PIV5 inhibits minigenome replication (23). An al-
ternative explanation for elevated viral gene expression in cells
infected with rPIV5-CPI? is that the V protein of rPIV5-
CPI? (Vcpi?) loses its ability to inhibit viral RNA synthesis.
To test this, we transfected plasmids encoding the V protein or
Vcpi? into the minigenome system together with plasmids
encoding the P protein or Pcpi-. We found that Vcpi? inhib-
ited minigenome replication (together with either the P pro-
tein or Pcpi-) as did the wt V protein (Fig. 2C and D), sug-
gesting that increased viral gene expression in cells infected
with rPIV5-CPI? is due to alterations in the P protein and not
to alterations in the V protein.
Gene expression from replication-defective minigenome sys-
tem. To investigate the mechanism for the more efficient
Pcpi? and Pcpi? proteins, we examined the effects of Pcpi?
and Pcpi? on viral RNA transcription. Previously we gener-
ated a minigenome system in which reporter gene expression
arises only from viral transcription, not replication, due to
mutation in the region that is essential for viral RNA replica-
tion (23). Using this transcription-only minigenome system, we
found that Pcpi-, Pcpi?, and Pwt all had similar abilities to
facilitate viral RNA transcription (Fig. 3), suggesting that the
increased activities of the Pcpi? and Pcpi? proteins comes
from a process other than transcription, i.e., viral RNA repli-
cation. This result is consistent with those obtained with virus-
infected cells (Fig. 1E, F, and G). Interestingly, overexpression
of the P protein, Pcpi?, or Pcpi? did not inhibit viral mRNA
transcription, suggesting that the inhibitory effect on viral gene
expression observed on overexpression of the P protein re-
ported previously (23) and observed earlier (Fig. 2A and B)
depends on viral RNA replication.
A single change at amino acid residue 157 of the P protein
is responsible for elevated viral gene expression. To further
identify the residue that plays a critical role in elevated viral
gene expression, we generated a series of P-protein mutations
that contain double or single amino acid residue changes at
positions 32, 33, and 157 (Table 1). Since the minigenome
system reproduces the observation made for virus-infected
cells concerning viral RNA synthesis and the minigenome sys-
tem is free of interference from the V protein, this system was
used to identify the amino acid residue that is responsible for
increased activity of the P protein. We found that whenever the
VOL. 82, 2008 ROLE OF PIV5 P PROTEIN IN VIRAL REPLICATION9127
FIG. 2. 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,
9128 TIMANI ET AL.J. VIROL.
amino acid residue at position 157 was changed from Ser to
Phe (S157F), the P protein had a higher activity than the wt P
protein (Fig. 4A), indicating that this residue plays a critical
role in modulating P-protein activity. The expression levels of
the different viral proteins were similar, as indicated by immu-
noblotting of the NP and P proteins (Fig. 4B; also data not
Protein-protein interactions involving the P protein. To fur-
ther investigate the mechanism of elevated viral RNA replica-
tion of Pcpi?, interactions between P proteins and other viral
proteins, as well as those between the P protein and itself, were
examined. It is known that the P protein interacts with the NP
protein to serve as a chaperone for the NP protein to encap-
sidate viral RNA genomes, and the P protein also interacts
with the L protein to stabilize the L protein. The P protein
forms a homo-oligomer, which is essential for its function. To
examine whether changes within the P proteins of Pcpi? and
Pcpi? affect their associations with the NP or L protein, co-
immunoprecipitation was performed using [35S]Met/Cys-la-
beled transfected cell lysates. The amounts of NP precipitated
by anti-P in Pcpi?transfected cells in Fig. 5A look different.
However, the expression level of Pcpi? is much lower than
those of the rest. When we adjusted the expression levels of P,
the amount of NP precipitated by anti-P in cells transfected
with Pcpi? is not obviously different from the rest. Thus, no
obvious differences were observed in interactions between NP
and P, Pcpi?, or Pcpi?. In addition, no obvious differences
were observed in interactions between L and P, Pcpi?, or
Pcpi? (Fig. 5A and B). It is unlikely that interactions between
P and NP or interactions between P and L play critical roles in
the increased activities of the Pcpi? and Pcpi? proteins. To
examine possible effects on P-protein oligomer formation, we
carried out cross-linking experiments. While there was a small
amount of the Pcpi? monomeric form in cross-linking exper-
iments, the amount was small and we did not consistently
observe it. In addition, the same thing was not observed with
Pcpi?. Because Pcpi? also caused elevated viral gene expres-
sion, we conclude that the small amount of the monomeric
form of Pcpi? is not significant for its ability to enhance viral
gene expression (Fig. 5C). Thus, it is unlikely that homo-oli-
gomer formation contributes to increased RNA replication
activities of Pcpi? or Pcpi?.
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.
FIG. 3. Transcription activities of P, Pcpi?, and Pcpi?. (A) A repli-
cation-deficient minigenome plasmid, pMG-m-R-Luc (23), was trans-
fected 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
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.
FIG. 4. Activities of mutant P proteins in minigenome system. (A) In-
creasing amounts of plasmids encoding P and different P mutant proteins
that contain double or single substitution mutations at amino acid posi-
tions 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, neg-
ative control which lacks L. Error bars are standard deviations of means
from examples containing six replicates for each transfection.
VOL. 82, 2008 ROLE OF PIV5 P PROTEIN IN VIRAL REPLICATION9129
Decreased phosphorylation of Pcpi? and Pcpi? proteins. It
is known that the P protein is heavily phosphorylated and its
phosphorylation plays a critical role in its function. The Pcpi?
protein contains three residues (V32I, T33I, and S157F) that
differ from the wt P protein, and two of them are potential
phosphorylation sites in the wt P protein (T33 and S157). To
determine whether these sites are involved in phosphorylation
of the P protein, cells were infected with mock, PIV5, rPIV5-
CPI? or rPIV5-CPI? and labeled with [35S]Met/Cys or
[33P]orthophosphate for 3 to 4 h at 1 dpi. The P proteins were
immunoprecipitated and resolved by SDS-PAGE. Interest-
ingly, both the Pcpi? and Pcpi? proteins had reduced phos-
phorylation levels (about 70% of that of the wt P protein) (Fig.
6A and B). That the reduction of phosphorylation in Pcpi? is
modest is consistent with the notion that the P protein is
phosphorylated at multiple sites and that these residues affect
phosphorylation only of a subset of these sites. Because rPIV5-
CPI? and rPIV5-CPI? have elevated viral gene expression,
the expression levels of P in these viruses are higher than that
in wt virus-infected cells.
S157 of P protein is phosphorylated. To investigate whether
T33 and S157 of the P protein are phosphorylated, we used a
web-based program, NetPhos (http://www.cbs.dtu.dk/services
/NetPhos/), that predicts phosphorylation sites within a pro-
tein. The program predicts that the likelihood of T33 being
phosphorylated is 0.025, while the likelihood of S157 being
phosphorylated is 0.994 (on a scale from 0 to 1, with 1 being the
highest likelihood of phosphorylation). Thus, it is predicted
that S157 is phosphorylated. To further investigate this possi-
bility, we purified the P protein from PIV5-infected cells using
affinity purification with anti-P antibody. The purified P pro-
tein was resolved by SDS-PAGE, and the P protein band was
excised and digested with trypsin. The digested peptides were
subjected to MS analysis for phosphorylation site determina-
tion. The MS detected 71% of the total P protein sequence. A
peptide containing S157 was identified in which the S157 po-
sition was indeed phosphorylated (Fig. 7). While peptides con-
taining T33 were identified in the MS analysis, no phosphory-
lation of this residue was detected.
To examine the role of phosphorylation at residue S157 in
viral RNA synthesis, we constructed P-protein S157A and
S157D mutants and examined the activities of these proteins
using the minigenome replication system (Fig. 8). As expected,
FIG. 5. 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 post-
transfection, the cells were metabolically labeled with
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 visu-
alized as described in Materials and Methods.
FIG. 6. 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 with35S-ProMix or [33P]orthophos-
phate 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 of33P/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).
9130 TIMANI ET AL.J. VIROL.
the S157A mutation resulted in higher reporter gene expres-
sion than that observed with the wt P protein. Interestingly, the
S157D mutation also resulted in higher reporter gene expres-
sion than that for the wt P protein.
In this work, we have investigated roles of the P and V
proteins in the phenotype of rPIV5-CPI?, which contains six
mutations in the shared region of the V and P proteins. One
main characteristic of rPIV5-CPI? is that it causes elevated
viral gene expression. Because the P protein plays an essential
role in virus RNA synthesis and the V protein regulates virus
RNA synthesis as well, it was not clear which of these altered
proteins contributes to the elevated viral gene expression. It is
possible that the P protein in rPIV5-CPI? (Pcpi?) gains new
function and/or the V protein in rPIV5-CPI? (Vcpi?) loses its
ability to inhibit viral RNA synthesis. We have found that the
Pcpi? protein indeed has elevated activity on viral gene ex-
pression. We have also found that the Vcpi? maintains its
ability to inhibit viral RNA synthesis in the minigenome sys-
tem. Thus, we conclude that the ability of rPIV5-CPI? to
cause elevated viral gene expression is likely due to a P protein
with increased ability to facilitate replication of viral RNA.
To study the mechanism of the elevated activity of the Pcpi?
protein, we examined viral RNA synthesis (viral RNA and viral
mRNA). We have found that rPIV5-CPI? causes elevated
viral RNA replication while maintaining similar levels of viral
mRNA transcription per viral genome. This result is consistent
with our observation that there is no difference between the
Pcpi? and P proteins in gene expression from the minigenome
system containing a defective trailer sequence in which only
viral transcription occurs, whereas Pcpi? enhances reporter
gene expression from a fully functional minigenome system.
Since we have not found any significant difference among P
FIG. 7. 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 yn-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.
FIG. 8. 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 de-
scribed in Materials and Methods. Neg, negative control without L.
Error bars are standard deviations of means from examples containing
six replicates for each transfection.
VOL. 82, 2008 ROLE OF PIV5 P PROTEIN IN VIRAL REPLICATION 9131
proteins in their ability to associate with NP or L and to form
homo-oligomers, we speculate that the elevated gene expres-
sion phenotype may be associated with a host protein. We have
mapped the amino acid residue that plays a critical role in the
elevated gene expression to serine at position 157. Interest-
ingly, S157 is phosphorylated in the wt P protein. Due to the
limitation of methods employed to determine phosphorylation,
it is not clear what percentage of P is phosphorylated, nor is it
known whether phosphorylation of S157 is dynamic and regu-
lated during the virus replication cycle. Nonetheless, this is the
first time a phosphorylation site has been identified in the P
protein of PIV5. Furthermore, this is first time that an effect on
viral gene expression has been observed after altering the
phosphorylation site of P in a paramyxovirus genome. Intrigu-
ingly, mutating this phosphorylation site to F results in higher
activity for the P protein, and this higher activity is limited to
viral RNA replication but not transcription, indicating that this
residue plays a critical role in modulating virus RNA replica-
tion. The exact mechanism for increased RNA replication with
the S157-to-F substitution is not clear. Interestingly, mutating
S157 to A or D also results in increased activity for the P
protein. It is noteworthy that the context of this S157 is within
a Polo-like kinase 1 (PLK1) binding motif (SpSP; the second S
is phosphorylated). We speculate that phosphorylation of res-
idue 157 of the P protein is important for its interaction with
PLK1 and that PLK1 may play an important role in regulating
viral RNA replication. Out of 10 strains of PIV5 examined, 7
have F residues at position 157 instead of the S residue found
in the common laboratory strain W3A (4). Further studies on
the role of PLK1 will address this interesting possibility.
This increased activity of the P protein may explain some
discrepancies in the literature. Previously a recombinant PIV5
lacking the conserved cysteine-rich C terminus (rPIV5V?C)
induced increased IFN-? and IL-6 expression, suggesting that
the V protein plays a role in inhibiting expression of IFN-? and
IL-6 in infected cells (15, 24). Ecotopic expression of the V
protein or the C terminus of the V protein is sufficient to
inhibit IFN-? expression (31). Thus, it is thought that the C
terminus is required and essential for the V protein to inhibit
IFN-? expression. Interestingly, it has also been reported that
rPIV5-CPI? induces expression of IFN-? (42). One of the
theories for explaining increased IFN-? expression by rPIV5-
CPI? is that the V protein (Vcpi?) in the rPIV5-CPI? virus
has lost its ability to inhibit IFN-? expression. However, be-
cause the mutations of Vcpi? are in the N terminus and the C
terminus of Vcpi? is still intact, the result is seemingly incon-
sistent with reports that the C terminus is required and suffi-
cient for inhibiting IFN-? expression. An alternative explana-
tion is that rPIV5-CPI? induces expression of IFN-?
differently from rPIV5V?C. Our result that Pcpi? is more
efficient in facilitating replication of viral RNA is consistent
with this hypothesis. It is possible that more efficient Pcpi?
protein has an advantage over the wt P protein in allowing
more rapid production of viral proteins in infected cells and
thus more virus progenies. However, the downside of this in-
creased replication rate may be increased or faster production
of viral proteins or RNA that can be detected by host cell
innate immune response sensors. This could conceivably lead
to enhanced production of IFN early in rPIV5-CPI? infec-
tions, before sufficient V protein has been produced to block
IFN signaling. Such a scenario is consistent with the observa-
tion that the V protein can inhibit IFN-? expression indepen-
dently of virus infection, such as double-stranded-RNA-in-
duced IFN-? expression (31). Recently it was reported with the
studies of rPIV5-CPI? that the P protein may have a role in
inhibiting host innate immune responses; however, this puta-
tive role of the P protein in limiting host innate immune re-
sponses has been observed only in virus-infected cells (9). In
this experimental system, when an additional copy of the P
gene was inserted into the genome of rPIV5-CPI?- (rPIV5-
CPI-/P), expression of IFN-? was reduced in the virus-infected
cells. It is possible that this additional copy of the P gene
reduces viral gene expression, since overexpression of the P
protein inhibits viral gene expression. That Pcpi? is more
efficient in facilitating replication of viral RNA is consistent
with the theory that synthesizing more viral RNA may result in
more-robust innate immune responses. However, the results
presented here do not exclude the possibility that the P protein
may be able to interrupt innate immune responses through a
mechanism that is independent of its role in viral RNA syn-
It is well known that overexpression of the P protein results
in inhibition of viral RNA synthesis, which is consistent with
our observation (Fig. 2 and 4) that using too much plasmid
encoding the P protein in the minigenome system reduces viral
RNA synthesis (23). Interestingly, increased expression levels
did not seem to inhibit reporter gene expression from the
minigenome system with a defective trailer sequence, in which
only viral RNA transcription is measured. Thus, it appears that
overexpression of the P protein inhibits viral RNA replication
while having no effect on viral RNA transcription. It is possible
that the overexpressed P protein may outcompete the NP pro-
tein, preventing it from encapsidating nascent viral RNA ge-
nomes, thus inhibiting viral RNA replication. In the case of
viral RNA transcription, overexpression of the P protein would
have no effect because the NP protein is not needed for func-
tionality of viral mRNA.
We thank the members of Biao He’s laboratory for helpful discus-
sion and technical assistance. We are grateful to Rick Randal for
providing antibody against P.
This work was supported by grants from the National Institute of
Allergy and Infectious Diseases to B.H. (AI051372 and K02 AI65795).
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