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Journal of Virology, July 2004, p. 7443-7454, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7443-7454.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of the Amino Acid Residues of Sendai Virus C Protein That Are Critically Involved in Its Interferon Antagonism and RNA Synthesis Down-Regulation

Atsushi Kato,1* Case Cortese-Grogan,2 Sue A. Moyer,2 Fumihiro Sugahara,3 Takemasa Sakaguchi,3 Toru Kubota,1 Noriyuki Otsuki,1 Masayoshi Kohase,1 Masato Tashiro,1 and Yoshiyuki Nagai4

Department of Virology 3, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo 208-0011,1 Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8551,3 Toyama Institute of Health, Kosugi-machi, Toyama 939-0363, Japan,4 Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, Florida 326102

Received 12 January 2004/ Accepted 25 February 2004


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ABSTRACT
 
Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.


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INTRODUCTION
 
Sendai virus (SeV) is an enveloped virus with a linear, nonsegmented, negative-sense RNA genome of 15,384 nucleotides and belongs to the genus Respirovirus of the subfamily Paramyxovirinae. SeV contains six genes in the order 3'-(leader)-N-P-M-F-HN-L-(trailer)-5' on the genome. The monocistronic mRNAs are transcribed by the viral RNA polymerase composed of L and P proteins (16). However, the P gene is exceptional in that it gives rise to multiple protein species by a process known as RNA editing and by the use of an overlapping open reading frame (ORF). The P protein is translated from the unedited mRNA, which is the exact copy of the P gene, while the V protein is translated from the edited mRNA, in which one nontemplated G residue is cotranscriptionally inserted to the editing position. The P and V proteins, therefore, have a common N terminus but have different C termini because the reading frame for V changes by +1 relative to the P frame (for a review, see references 33 and 38). The V protein is encoded by almost all viruses that are members of the subfamily Paramyxovirinae. The unique C-terminal region of V contains seven cysteine residues highly conserved among paramyxoviruses, forms zinc finger-like motifs, and, indeed, binds Zn2+ (7, 20, 35, 44, 51).

The C protein is translated from the –1 reading frame relative to the frame common to P and V. The SeV P and V proteins are initiated at the AUG codon at position 104, whereas the SeV C reading frame produces a nested set of C', C, Y1, and Y2 proteins initiating, respectively, at a non-AUG (ACG) codon at position 81 and at AUG codons at positions 114, 183, and 201 (4, 15, 45). The C', C, Y1, and Y2 proteins are terminated at the same position 726 and are collectively called C proteins. Among them, the C protein is the major species expressed in infected cells, at a molar ratio severalfold higher than that of the other three proteins (32). The C proteins are expressed by the viruses belonging to three genera, Respirovirus, Morbillivirus, and Henipahvirus, and by Tupaia paramyxovirus-like viruses, but they are not expressed by the viruses belonging to two genera, Rubulavirus and Avulavirus of Paramyxovirinae (reviewed in reference 37). The amino acid sequence of the C proteins is well conserved within each genus but poorly conserved (less than 20%) between different genera. The C proteins are all relatively small (150 to 220 amino acids [aa]) and highly basic, with an isoelectric point of around 10 (38).

Both the V and C proteins of SeV are categorized as accessory proteins that may not always be essential for the viral life cycle, because there is at least one virus which does not have or does not express these proteins in its close relatives (the genus Respirovirus) (33). We have employed two complementary approaches, SeV reverse genetics to delete the V and C proteins and plasmid-based expression of these proteins, to address how these proteins contribute to actual viral replication and pathogenesis (reviewed in references 37, 38, and 39). The C proteins are indeed dispensable for SeV replication in cultured cells, but the lack of C proteins profoundly affects the viral life cycle. Compared with the wild type, mutant SeV with the four C proteins (C'/C/Y1/Y2) knocked out produced nearly 1/104 of the progeny virus in ovo, 1/103 in tissue cultured cells, and an undetectable level in mouse lungs (32). The SeV C protein is extremely versatile. The first finding about the role of SeV C proteins involves the inhibition of viral RNA synthesis; C proteins supplied from the plasmid inhibit minigenome RNA synthesis in a dose-dependent manner in vitro (3). A larger quantity of viral RNA is synthesized in cells infected with the four-C knockout SeV than in the parental wild-type SeV-infected cells at the late stage of infection (17). Moreover, all the C-, Y1-, or Y2-expressing cell lines suppress SeV multiplication at the transcriptional level (24). Analysis of the stable transformants constitutively expressing various C-truncates from the N and C termini indicated that the 106-residue C-terminal half of the C proteins is sufficient for viral RNA inhibition (23). In the cells constitutively expressing SeV C protein, SeV growth is significantly suppressed because of the inhibition of viral RNA synthesis. This growth inhibition by SeV C proteins is also found in closely related human parainfluenza virus type I (hPIVI) but not in the more distantly related hPIV3 and measles virus. Based on these findings, C proteins are thought to down-regulate viral RNA synthesis specifically. This inhibition is suggested to occur via binding of the C proteins with the L protein (14, 19).

The second finding about the role of SeV C proteins concerns the promotion of viral assembly and/or budding. SeV C proteins are expressed abundantly in infected cells but are incorporated in trace amounts in the virions and, thus, are practically considered to be nonstructural proteins having nothing to do with virion assembly (34, 46). However, as described above, production of the progeny viruses was hampered greatly in the cells infected with the four-C knockout SeV even enough viral genomic RNA and proteins were accumulated in the cells. More surprisingly, in the culture supernatant of cells infected with the SeV mutant with the four C proteins knocked out, there were many noninfectious particles with a sedimentation profile distributing widely from light to heavy fractions in the sucrose gradient centrifugation and with a highly anomalous morphology in the electron microscope observation (17). The role of the C proteins in this process remains to be clarified.

The third finding about the role of SeV C proteins is that they counteract the antiviral activity of interferons (IFNs). Studies performed in the 1960s showed that preinfection or persistent infection of cells with SeV or hPIV3 enhanced the growth of heterologous postinfecting IFN-sensitive viruses, such as Newcastle disease virus and vesicular stomatitis virus (VSV), suggesting that preinfection with SeV and hPIV3 somehow rendered cells unresponsive to autocrine IFNs (18, 36). The anti-IFN capacity of these viruses was recently established (5, 13). HeLa cells preinfected with the parental wild-type and C'/C-, C'/C/Y1-, and V-knockout SeVs circumvented the anti-VSV action of IFN-ß, whereas the virus with the four C proteins knocked out lost this IFN antagonism (13). The C-, Y1-, or Y2-expressing cell lines were all capable of circumventing the activation of IFN-stimulated genes (ISGs) and the induction of an antiviral state by IFN-{alpha} or -ß and IFN-{gamma} (24). These observations suggested that IFN signaling was somehow blocked by the SeV C', C, Y1, or Y2 protein without the aid of other viral proteins. An analysis of the stable transformants expressing various truncates from the N and C termini indicated that the 106-residue C-terminal half of C protein was sufficient for IFN antagonism (23). The C protein with a spontaneous mutation at residue 170 of phenylalanine to serine (CF170S) (22), within the 106-residue region, neither antagonized IFNs (10) nor, compared to the effect of the parental C in vitro, inhibited viral RNA synthesis (14). IFN antagonism is fully generated even in cells expressing trace amounts of the C proteins, in which the inhibition of viral RNA synthesis is no longer observed.

The fourth finding about the role of the SeV C proteins is that they suppress apoptosis. The parental wild-type SeV grows without any significant cytopathic effect in Hep-2 cells, and infected cells tolerate the apoptotic stimulus caused by VSV (29). Though cells infected either with C'-, C/C'-, or C'/C/Y1-knockout viruses tolerate the apoptosis signals as do the wild-type SeV-infected cells, cells infected with the four-C knockout SeV clearly showed the condensation of nuclei and the fragmentation of chromosomal DNA, indicating that SeV C proteins had antiapoptotic activity (28).

In this report, we attempted to determine which regions of the SeV C protein were responsible for IFN antagonism and for the down-regulation of viral RNA synthesis by using cells expressing the wild-type C protein and several mutant C (Cm) proteins with charged amino acid substitutions for alanine residues, most of which were within the C-terminal half. A substitution at positions 151, 153, and 154 of the C protein abolished the anti-IFN and RNA synthesis-inhibiting activities simultaneously, but all of the other substitutions except the mutation at positions 114 and 115 abolished only the RNA synthesis-inhibiting activity. From these results, we conclude that the amino acids scattered throughout the C-terminal half of the C protein govern the down-regulation of viral RNA synthesis, while more limited residues are critical for playing a role in IFN antagonism. Using the various Cm proteins, we further found that SeV C proteins participate in the inhibition of IFN signaling through the unusual phosphorylation and dephosphorylation of STAT1 and STAT2, which play key roles in an IFN-{alpha}/ß system.


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MATERIALS AND METHODS
 
Plasmids and stable transformants. A plasmid, pKS-C, expressing C protein but not Y1 or Y2, was described previously (24). The plasmids encoding the mutated C proteins pTM1gstC'2, pTM1gstC'3, pTM1gstC'4, pTM1gstC'5, pTM1gstC'6, and pTM1gstC'8 (14), all with charged amino acids substituted for alanine residues, were used as the PCR templates to obtain a DNA fragment with the suitable restriction enzyme sites in both termini. Briefly, two primers, CF (5'-GAATTCHindIIIAAGCTTGCC114ATGCCTTCATTCTTAAAG-3' ) and CR (5'-GAATTCBamHIGGATCCCTA726TTACTCTTGCACTATGTG-3' ), were used for the amplification. In the primer sequences, the initiation and termination codons are underlined and the positions from the cap site of P/V mRNA are numbered (see below for restriction sites). A three nucleotide-region (italicized) upstream of the initiation codon was modified to optimize for the translation according to Kozak's rule (30).

DNA fragments corresponding to V, V/P common, and V unique regions were prepared by PCR using a template, pSeV+-4C–, which has disrupted ORFs of all of the four C proteins without changing an overlapping P ORF (32). Two primers, VF (5'-TGAGHindIIIAAGCTTGGTACC104ATGGATCAAGATGC-3') and V/PR (5'-ATCGATBamHIGGATCCTTAGCCCTTTTTTGTTGA-3' ), were used for the amplification of the 5' terminal half of the V ORF encoding the V/P common domain. For the V ORF, fragments of the 5' half and 3' half were individually amplified by using primers to insert a G residue at the editing site of the genomic cDNA ORF. The primers VF and Vedit (5'-GACTCAACAAAAAAGGGGCATAGGAGAGAA-3') were used for the 5' half of the fragment, and VeditC, a complementary primer of Vedit, and VR (5'-ATCGATBamHIGGATCCTTACGAGCGGAAGATTTC-3' ) were used for the 3' half of the fragment. The two fragments were purified and mixed and were further combined at the overlapping site by PCR using both termini of primers VF and VR. For the V unique domain, fragments were amplified by PCR with primers MycF (5'-TGAGHindIIIAAGCTTGGTACCATGGAGCAGAAGC-3') and VR. In the primer sequences, the initiation and termination codons are also underlined (see below for restriction sites).

These fragments were cut with HindIII and BamHI (shown as superscripts in the primers) and cloned into the same sites of the pKS336 plasmid (GenBank accession number AF403737). The plasmids encoding C proteins with the charged amino acids substituted for alanine residues at the indicated positions were as follows: 77 and 80 (pKS-Cm2); 114 and 115(pKS-Cm3); 139 and 142 (pKS-Cm4); 151,153 and 154 (pKS-Cm5); 156 (pKS-Cm6); and 173, 175, and 176 (pKS-Cm8). Each position was numbered from the first amino acid of the C protein. The plasmids encoding the V, V/P common, and V unique regions were named pKS-V, pKS-V/P, and pKS-Vu, respectively. Furthermore, the plasmid encoding the mutant V protein with the two substitutions in the cysteine residues (C362S and C365R) was named pKS-Vcys. This mutation reduced zinc-binding capacity to 30% and impaired virus virulence to mice to 1/25 (7, 20). After the verification of the sequences, stable transformants expressing the designed proteins were established as described previously (24).

Immunoprecipitation. Parental HeLa cells and the wild-type or mutated C- or V-expressing HeLa cells were inoculated to a density of 5 x 105 cells per plate in the 35-mm plates 1 day before the labeling. The next day, cells were washed once with phosphate-buffered saline (PBS) and replaced in 0.5 ml of Dulbecco's modified Eagle's medium (DMEM) without methionine. After 30 min for the starvation, 0.74 MBq (20 µCi) of L-[35S]methionine (43.48 TBq/mmol; ICN Inc., Aurora, Ohio) per plate was added. Cells were incubated at 37°C for 2 h in 5% C02 and then washed twice with ice-cold PBS. The harvested cells were then lysed with 0.5 ml of radioimmunoprecipitation assay buffer on ice for 5 min and spun at 14,000 x g for 5 min at 4°C. Immunoprecipitations were performed as described previously (26), and the precipitates were analyzed on a 4 to 12% acrylamide-Bis-Tris gradient gel (NuPAGE; Invitrogen Corp., Carlsbad, Calif.), which was subsequently treated with Enlightning reagent (Perkin Elmer Life Sciences NEN, Boston, Mass.) for 30 min, dried, and visualized by fluorography.

Western blotting. Cytoplasmic cell extracts in radioimmunoprecipitation assay buffer were run through 4 to 12% acrylamide-Bis-Tris gels (NuPAGE; Invitrogen Corp.) and electroblotted onto hybond enhanced chemiluminescence nitorocellulose membranes (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). The membranes were blocked for 1 h in 3% skim milk in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 and probed with anti-STAT1{alpha}/ß (sc-346; Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-pY-STAT1 (9171; Cell Signaling Technology, Beverly, Ma.), anti-STAT2 (sc-476; Santa Cruz Biotechnology), anti-pY-STAT2 (07-224; Upstate Biotechnology, Lake Placid, N.Y.), anti-PKR (sc-707; Santa Cruz Biotechnology), or anti-SeV C serum. The viral proteins synthesized in the cells were probed with anti-SeV or anti-VSV serum, which was kindly supplied by B. Gotoh (Fukui School of Medicine). Immunoreactivity was detected by the peroxidase-based electrochemiluminescence method using an enhanced chemiluminescence detection reagent (RPN2109; Amersham Pharmacia Biotech) according to the manufacturer's instructions. The reacted bands on the membranes were detected and incorporated digitally by a luminescent image analyzer (LAS-1000 plus; Fuji Photo Film Co., Ltd., Tokyo, Japan). The dendrograms of each band were achieved by using the National Institutes of Health Image software and were used for calibrating the relative intensity.

Antiviral activity of IFNs. Parental HeLa cells and the wild-type or mutated C- or V-expressing HeLa cells plated at a density of 5 x 104 cells per well in 24-well plates were treated with human IFN-ß or IFN-{alpha} at a concentration of 0, 10, 100, or 1,000 IU/ml for 24 h in serum-free DMEM. The cells were washed once with PBS and infected with VSV at a multiplicity of infection of 2 per cell in 0.1 ml of serum-free DMEM for 1 h at 37°C. After removal of the virus-containing media, cells were further incubated in the same medium without IFN until significant cytopathic changes developed in the parental HeLa cells (12 to 24 h). For viral cytopathic effect assays, cells were fixed and stained with Amid Black 10B.

Reporter gene-expressing activity of rSeV. The strain Z of recombinant SeV harboring the luciferase gene (rSeV/luci) was inoculated into parental HeLa cells and the wild-type or Cm-expressing HeLa cells at a multiplicity of infection of 5 per cell in a 6-well plate (25). The infected cells were maintained in serum-free DMEM. The luciferase activities and the protein quantities in rSeV/luci-infected cells were measured at 0, 2, 4, and 6 h postinfection by using a dual luciferase assay kit (Promega, Madison, Wis.) and a protein assay kit (Bio-Rad Laboratories, Hercules, Calif.), respectively.

Sucrose density-gradient centrifugation of cell lysates. Cells (107 in a 175-cm2 tissue culture bottle) were harvested from the culture bottle by trypsin and collected by centrifugation at 270 x g for 10 min. The pellets of cells were lysed on ice with 2 ml of lysis buffer (10 mM sodium phosphate [pH 7.2], 100 mM NaCl, 1% Triton X-100). The resulting cell lysates were centrifuged at 3,000 x g for 15 min to pellet nuclei and cellular debris. One milliliter of supernatants was layered on the top of a 5 to 35% sucrose density gradient in 10 mM sodium phosphate (pH 7.2), 100 mM NaCl, and 0.1% Triton X-100 and ultracentrifuged with an SW42 (Beckman Coulter Inc., Palo Alto, Calif.) rotor at 38,000 rpm for 16 h at 4°C. The gradients were fractionated from the top to bottom of the tube into 16 fractions of 0.75 ml each.


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RESULTS
 
Anti-IFN activity of SeV accessory proteins. In previous studies it has been shown that SeV C proteins have the ability to inhibit the signal transduction of IFNs (13, 24). On the other hand, many viruses belonging to the subfamily Paramyxovirinae have been shown to antagonize IFN(s) via the activity of the V protein. Among them, simian virus 5 (5, 6), mumps virus (31), hPIV2 (40, 42), and Newcastle disease virus (21, 43) do not have the C protein. Even in the measles virus (41, 52, 54) and Nipah virus (47), which have both C and V proteins, a contribution of V protein to IFN antagonism has been reported. The unique C terminus of V proteins is the most conserved protein in the paramyxoviruses and is reported to be required for the antagonism of IFN in simian virus 5 (2), mumps virus (31), and hPIV2 (40). We therefore revisited the SeV V protein to determine whether or not it can antagonize IFNs. Four HeLa cell lines expressing the entire SeV V protein, the V protein with a modified cysteine motif (Vcys), the V unique C terminal region (Vu), or the V/P common region were established (Fig. 1A). The V and Vcys proteins could be immunoprecipitated by both anti-V and anti-P sera. The Vu protein could be precipitated by the anti-V serum but not by the anti-P serum, while the V/P protein could be precipitated by the anti-P serum but not by the anti-V serum. The expression levels of these proteins were almost comparable to that of V protein in the early stage (<12 h) of growth of SeV-infected cells.



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  		FIG. 1. Establishment of HeLa cell lines expressing the native, mutated, and truncated forms of the SeV V protein and their responses to exogenously added IFN-ß. (A) The HeLa cell lines expressing the SeV native V protein of 384 aa, P/V common region of 316 aa, V unique frame region of 68 aa (Vu), and a mutant V protein (Vcys) in which two of the seven cysteine residues at positions 362 and 365 were replaced by serine and arginine, respectively, are generated. The individual protein expressions were confirmed by immunoprecipitation by anti-V and anti-P sera. (B) Each cell line was pretreated with various amounts (0 to 103 IU/ml) of IFN-ß for 24 h and then challenged with VSV or left unchallenged (Mock). Anti-VSV action is assessed by the presence of cells attached to the plates. C, cells stably expressing the SeV C protein; None, the parental HeLa cell line expressing none of the SeV proteins.

To observe the effect of V protein and its derivatives on the establishment of an antiviral state, cells preincubated with IFN-ß at a concentration of 0, 10, 100, or 1,000 IU/ml for 24 h were challenged with VSV. As shown in Fig. 1B, IFN-ß alone did not cause any apparent cytopathogenicity in any cells tested under these conditions. All of the cells cultured in the absence of IFN-ß were detached from the plates by subsequent VSV infection. The parental HeLa cells were protected from the VSV-induced cytopathic effect by IFN-ß pretreatments at concentrations of 100 and 1,000 IU/ml. In contrast, HeLa cells expressing SeV C protein were totally detached by VSV infection even after pretreatment with IFN-ß at a concentration of 1,000 U/ml, as indicated previously (24). However, all cells expressing V, Vcys, Vu, or V/P were protected from VSV infection at IFN-ß concentrations of 100 and 1,000 U/ml, as seen in the parental HeLa cells. These results confirmed that it was the SeV C protein, but not the V protein, that could antagonize the IFN-ß. However, the possibility that the SeV V protein would enhance IFN antagonism in combination with the C protein was not excluded.

Establishment of cell lines expressing the C proteins with the charged amino acid-to-alanine replacements. We demonstrated previously that not only the C protein but also the smaller Y1 and Y2 proteins had the ability to both counteract the antiviral effect of IFNs and inhibit viral RNA synthesis (24). Furthermore, the regions responsible for conferring these abilities were mapped to the carboxyl terminal 106-aa region (residues 99 to 204) of the C protein, named Y3, which was common to all C proteins (23). To identify the amino acids in the C protein that are important for counteracting IFNs and for inhibiting viral RNA synthesis, the DNA fragment encoding substitutions of charged amino acids for alanine residues in Cm proteins, which were previously shown to have various L-binding activities and RNA synthesis-inhibiting activities in vitro (14), were subcloned into a mammalian expression vector and used to obtain HeLa cell transfectants that constitutively expressed each Cm protein.

The resulting HeLa cell lines were named Cm2, Cm3, Cm4, Cm5, Cm6, and Cm8, with substitutions of charged amino acids for alanine residues at the following positions, respectively, in the C protein: 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; and 173, 175, and 176. Each of the mutated proteins could be immunoprecipitated with anti-SeV C serum after the pulse radiolabeling (2 h) and could also be detected by immunoblotting with the same antibody (Fig. 2A). The amount of Cm protein expressed differed among the cell lines, probably due to the number of plasmids integrated into the chromosome, but did not differ in the same cell lines between the two detection methods, i.e., immunoprecipitation of pulse-labeled protein or immunoblotting of accumulated protein. These results indicated that the intracellular stability of each Cm protein did not change dramatically by the respective charged residue-to-alanine replacements.



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  		FIG. 2. Establishment of HeLa cell lines expressing C protein mutants with a series of charged amino acid substitutions for alanine residues and their responses to IFN-ß. (A) Cells expressing various Cm (Cm2 to Cm8) proteins were established. The expression of individual proteins was confirmed by both immunoprecipitation of the pulse-labeled cell lysates (top) and Western blotting (bottom). (B) The individual cell lines were compared for their responses to IFN-ß by using VSV as a challenge virus as described in the legend of Fig. 1B. (C) Antiviral activities of IFN-ß (103 IU/ml) in each cell line were analyzed by the inhibition of synthesis of VSV-specific N, P, and M proteins. C, cells stably expressing the wild-type C protein; None, the parental HeLa cell line expressing none of the SeV proteins.

Anti-IFN activity of mutated C proteins. As shown in Fig. 2B and C, the parental HeLa cells were protected from the VSV challenge by pretreatment with 100 or 1,000 unit of IFN-ß per ml, and no VSV proteins were detected in the cells pretreated with 1,000 U of IFN-ß per ml by immunoblotting with anti-VSV serum. On the other hand, the cells expressing the wild-type C protein detached from the bottom of the culture dish irrespective of IFN pretreatment, and the VSV proteins were clearly detected in the cells by immunoblotting. The cells expressing the Cm2, Cm3, Cm4, Cm6, and Cm8 proteins allowed the growth of VSV and detached from the culture plate even after treatment with IFN-ß at a concentration of 100 or 1,000 U/ml, just as did the cells expressing the wild-type C. Even though their expression levels were lower compared with the levels of the other Cm proteins (Fig. 2A), Cm4 and Cm6 were almost fully capable of antagonizing IFN (Fig. 2B and C). On the other hand, in cells expressing the Cm5 protein, IFN was fully active, greatly inhibiting VSV growth and allowing the cells to remain attached to the plates even with treatment of 100 or 1,000 U of IFN-ß per ml (Fig. 2B). This indicated that Cm5 lost the capacity for anti-IFN activity. Essentially the same results were obtained when IFN-{alpha} was used instead of IFN-ß (data not shown).

Effect of mutations of SeV C protein on IFN-ß-induced signaling. To clarify the effect of mutated C proteins on IFN-ß-mediated signaling, the levels of the ISG products, STAT1{alpha}/ß, STAT2, and PKR, and the IFN-{alpha}/ß-responsive luciferase reporter gene expression were examined. In the parental HeLa cells, intracellular levels of STAT1{alpha}/ß, STAT2, and PKR (Fig. 3A) and IFN-stimulated response element (ISRE)-luciferase reporter gene activities (Fig. 3B) were increased in accordance with the hours of incubation with IFN-ß. Little or no stimulation of STAT1{alpha}/ß, STAT2, and PKR synthesis or of ISRE-reporter gene expression was found in cells expressing C, Cm2, Cm3, Cm4, Cm6, or Cm8. However, in Cm5-expressing cells, appreciable stimulations of STAT1{alpha}/ß, STAT2, and PKR synthesis and of ISRE-reporter gene expression were observed, indicating that the substitutions at positions 151, 153, and 154 resulted in the failure to block the IFN-ß-mediated signaling. Thus, the results of this signaling assay and of the bioassay (anti-VSV action) shown in the previous section were in very good agreement with each other and suggested that relatively limited amino acid residues were critically involved in IFN antagonism of the SeV C protein.



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  		FIG. 3. Expression of IFN-responsible genes in the parental and C-expressing HeLa cells. Induction of ISG products, STAT2, STAT1{alpha}/ß, and PKR (A) and stimulation of ISRE-driven luciferase gene expression (B) are shown in cells expressing the various Cm proteins. These were assayed as described in Materials and Methods and were examined after treatment of the cells with 1,000 IU of IFN-ß per ml for 0 to 8 h. C, cells stably expressing the wild-type C protein; None, the parental HeLa cell line expressing none of the SeV proteins. Relative luciferase activities (solid bars) are indicated as the proportional percentage of the luciferase value (counts per minute) per microgram of protein of C-expressing cells at 0 h.

Anti-IFN activity and STAT1 binding. Several studies have reported a good correlation between anti-IFN activity and the physical association of SeV C with STAT1 (8, 12). And, indeed, in our stable cell lines expressing C, Y1, or Y2 protein, anti-STAT1 antibody could precipitate STAT1 and C, Y1, or Y2 protein simultaneously (Fig. 4A). The binding of SeV Cm proteins with STAT1 was then studied in the context of anti-IFN activity. As shown in Fig. 4B, anti-C antibody could precipitate both C and STAT1, and, conversely, anti-STAT1 could precipitate STAT1 and C protein. In the parental HeLa cells, no STAT1 was precipitated by anti-C serum, and no C protein was precipitated by anti-STAT1 serum. These results showed the specificity of the immunoprecipitation procedure. However, none of the Cm proteins could be coprecipitated with STAT1 by anti-STAT1 serum, and, conversely, STAT1 could not be coprecipitated with any of the Cm proteins by anti-SeV C serum, irrespective of its anti-IFN activity. Even though the Cm2, Cm3, Cm4, Cm6, and Cm8 proteins could antagonize IFNs, they seemed not to bind with STAT1.



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  		FIG. 4. Association of SeV C protein with STAT1. The parental cells and cell lines expressing the wild-type and Cm proteins were pulse-labeled with [35S]methionine, and their lysates were immunoprecipitated with anti-STAT1 or anti-SeV C antiserum (A and B). The STAT1 precipitated with anti-C and the C protein precipitated with anti-STAT1 are marked with an asterisk (*) in panel B. The lysates from cells expressing C, Cm2, Cm3, and Cm5 were subjected to 5 to 35% sucrose gradient centrifugation, and the fractions (4 to 16) were immunoblotted either with anti-STAT1 or anti-SeV C serum (C). The formation of HMWCs between C or Cm proteins with STAT1 would be indicated by the sedimentation of these proteins in lower gradient fractions (14 to 16) and was observed only for wild-type C.

To confirm the inability of Cm proteins to bind with STAT1, an additional method was used. The cells expressing C, Cm2, Cm3, and Cm5 were lysed, centrifuged to remove their nuclei, and fractionated by 5 to 35% sucrose gradient centrifugation. The recovered fractions were then separated electrophoretically and immunoblotted with anti-STAT1 and anti-SeV C serum (Fig. 4C). In the C-expressing cells, STAT1 and C proteins were found both in the light (fractions 4 to 6) and in the heavy (fractions 14 to 16) fractions. The STAT1 and C proteins in the light fractions could represent soluble free molecules, and those in the heavy fractions could be involved in a high-molecular-weight complex (HMWC) (53). Detectable amounts of STAT1 and C proteins were also cosedimented into the intermediate fractions, indicating that several different forms of complexes might be generated between STAT1 and SeV C proteins.

On the other hand, in Cm5-expressing cells, which lost anti-IFN activity, STAT1 and C proteins appeared only in the light fractions (fractions 4 and 5) and no Cm5 protein or STAT1 sedimented in the heavier fractions, indicating little association of Cm5 with STAT1. These results support the previous finding that a C mutant (CF170S), which did not have anti-IFN activity, lost the ability to bind with STAT1 (10). However, in both Cm2- and Cm3-expressing cells, which were representatively used as the mutants retaining anti-IFN activity, STAT1 and the Cm proteins appeared only in the light fractions (fractions 4 and 5). Neither Cm2 nor Cm3 protein nor STAT1 sedimented into the heavier fractions (Fig. 4C). These results obtained by immunoprecipitation (Fig. 4B) and by sedimentation (Fig. 4C) suggest that the formation of HMWCs or of stable complexes of C proteins with STAT1 are not always required for the anti-IFN activity of SeV C protein.

Anti-IFN activity and the phosphorylation of STAT1 and STAT2. IFN-{alpha}/ß signaling is mediated through the JAK-STAT pathway (for a review, see references 1 and 50). Briefly, binding of IFN-{alpha}/ß to a cell surface IFN-{alpha}/ß receptor activates the receptor-bound kinases JAK1 and Tyk2, which subsequently phosphorylate the tyrosine residue of STAT1 and of STAT2 at positions 701 and 690, respectively. The tyrosine-phosphorylated (pY)-STAT1 and pY-STAT2 are required for forming a heterodimer and translocation to the nucleus, where they bind with IRF-9 and work as the transcriptional activator of ISGs (11, 49, 50). Subsequently, pY-STAT and pY-STAT2 are dephosphorylated in the nuclei to down-regulate the ISG transcriptions.

In the parental HeLa cells exposed to IFN-ß, pY-STAT1 and pY-STAT2 reached a peak within 60 min, and then pY-STAT1 decreased quickly at around 240 min posttreatment, while pY-STAT2 decreased gradually and was still detectable at 480 min posttreatment (Fig. 5). Under these conditions, the levels of nonphosphorylated STAT1 and STAT2 increased continuously for at least 480 min. In the C-expressing cells, which did not respond to IFNs, the level of pY-STAT1 increased continuously at least up to 480 min posttreatment, whereas pY-STAT2 was not detectable at any of the time points used. Since the levels of STAT1 and STAT2 did not significantly change in C-expressing cells during the incubation period, the continuous presence and increase of pY-STAT1 can be explained by the increased protein phosphorylation and/or decreased protein dephosphorylation, and the disappearance of pY-STAT2 can be explained by the negligible or absent STAT2 phosphorylation or its increased dephosphorylation in the C-expressing cells. In Cm5-expressing cells, which could respond to IFNs, the developmental sequences of pY-STAT1 and pY-STAT2 were essentially the same as in the parental HeLa cells. In Cm2-, Cm3-, and Cm8-expressing cells, which could not respond to IFNs, the continuous presence of pY-STAT1 and the total disappearance of pY-STAT2 were essentially the same as found in the C-expressing cells. Thus far, the C mutant proteins retaining IFN antagonism showed the phosphorylation and dephosphorylation abnormality of STAT1 and STAT2.



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  		FIG. 5. Effects of the SeV C protein on tyrosine phosphorylation of STAT1 or STAT2. Parental and the various C-expressing HeLa cells were treated with 1,000 IU of IFN-ß/ml for the indicated time (min). The cell lysates were immunoblotted with anti-STAT1, anti-phospho-tyrosin701-STAT1 (pY-STAT1), anti-STAT2, or anti-phospho-tyrosin690-STAT2 (pY-STAT2).

Effect of mutated C proteins on viral RNA synthesis. The inhibition of viral RNA synthesis in cells expressing the various Cm proteins was examined in the context of viral infection by using the recombinant SeV, rSeV/luci, containing the firefly luciferase gene as a reporter gene. rSeV/luci was inoculated into the parental and transfected HeLa cells, the latter expressing the wild type or each mutated C protein. Cell lysates were prepared at 0, 2, 4, and 6 h postinoculation for measuring the luciferase activity and the protein quantity. Potential fluctuations in the cell amounts were normalized by the protein quantity of cell lysate. The reporter luciferase activity in rSeV/luci-infected cells was well correlated with the amount of luciferase mRNA in the early stage (<12 h) of infection (25), and, indeed, reporter luciferase activity increased linearly and dramatically in parental HeLa cells at least up to 6 h postinfection (Fig. 6A). In contrast, the activities of C protein-expressing cells were lower than that of the parental cells at every compared time point, indicating the inhibition of SeV mRNA synthesis by SeV C proteins. In Cm3-expressing cells, the reporter luciferase activities were as strongly inhibited as in the C protein-expressing cells, but in the other five Cm protein-expressing cell lines, i.e., Cm2, Cm4, Cm5, Cm6, and Cm8, the reporter expressions were scarcely inhibited.



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  		FIG. 6. Inhibition of viral gene expressions by various SeV C proteins. Reporter luciferase gene expression from the infecting recombinant SeV (A) and the expression of the SeV P and N proteins (B) were examined at the indicated hours. The SeV P and N proteins produced in the infected cells were visualized by immunoblotting with anti-SeV serum. C, cells expressing the wild-type C protein; None, the parental HeLa cell line expressing none of the SeV proteins. Relative luciferase activities (solid bars) are indicated as the luciferase value (counts per minute) per microgram of protein in SeV/luci-infected cells.

The inhibition of viral RNA synthesis was reconfirmed by comparing SeV proteins accumulated in the parental cells with those in the individual Cm-expressing cells. The cell lysates prepared at 6 h postinfection were immunoblotted by anti-SeV serum. SeV N and P proteins were clearly detected in the parental cells but were detected very weakly in cells expressing C and Cm3 (Fig. 6B). This indicated that the synthesis of both the luciferase and SeV proteins expressed from the rSeV/luci genome was strongly restricted in cells expressing SeV C or Cm3 protein. In contrast, in cells expressing Cm2, Cm4, Cm5, Cm6 and Cm8, the amounts of accumulated SeV proteins were almost equivalent to those in the parental cells (Fig. 6B), in good agreement with the data indicating that the Cm2, Cm4, Cm5, Cm6, and Cm8 proteins greatly reduced their RNA synthesis-inhibiting activities (Fig. 6A).

Quantitative analysis of RNA synthesis inhibitions. The inhibition of viral RNA synthesis required a considerably high level of intracellular quantity of the C protein (3). In the above studies, cells expressing Cm2, Cm4, Cm5, Cm6, and Cm8 were found to hardly inhibit SeV RNA synthesis. However, the intracellular levels of some mutants such as Cm4 and Cm6 were obviously lower than those of the others or the wild-type C (Fig. 2A). Thus, there was clearly a need for more quantitative analyses to reach a conclusion regarding the anti-RNA synthesis activity of the various C mutants. The expression levels of C proteins shown in Fig. 2A were quantitatively measured by image-analyzing software, and the relative expression levels were indicated by standardizing the wild-type C quantity as 100. The relative expression levels were calibrated to be 95 (Cm2), 115 (Cm3), 29 (Cm4), 93 (Cm5), 40 (Cm6), and 57 (Cm8) and are plotted (Fig. 7A, x axis). The relative inhibition value of RNA synthesis was calculated by using the reporter luciferase activities from the rSeV/luci at 6 h postinfection by standardizing the activities in C cells as 100 and in the parental HeLa cells as 0. The relative inhibition values were calibrated to be 34 (Cm2), 124 (Cm3), 0 (Cm4), 14 (Cm5), 24 (Cm6), and 17 (Cm8) (Fig. 7A, y axis). When the value ([x, y]: relative expression level, relative inhibition level), of parental cells (0,0) was connected with that of C cells (100, 100), the values of the Y1 and Y2 proteins stably expressed in HeLa cells (including three other independent clones, Y2a, Y2b, and Y2c) as established in a previous study (24) and of Cm3 in the present study could be plotted at points very close to this diagonal line (Fig. 7A). These results clearly showed that the Cm3 protein retained the inhibiting activity as did the wild-type C, Y1, and Y2. In contrast, the Cm2, Cm5, and Cm8 proteins appeared to have greatly reduced RNA synthesis-inhibiting activities, because they were located well below the line.



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  		FIG. 7. Relationships between SeV RNA synthesis inhibition and the expression levels of Cm proteins. (A) The SeV RNA synthesis inhibition levels and intracellular levels of various C proteins including Y1 and Y2 were calibrated by using the luminescent image analyzer and National Institutes of Health Image software. (B) Four additional clones of Cm4 (Cm401, Cm402, Cm403, and Cm406)- and Cm6 (Cm606, Cm607, Cm609, and Cm610)-expressing cells were established, and the actual intracellular levels of the mutant proteins (top) and luciferase expression from the rSeV in these at 2, 4, and 6 h indicated (bottom). (C) Based on the data shown in B, the relationships between the RNA synthesis inhibition and C protein levels were analyzed as in A. See Results for an explanation of the standardization of values.

However, in the case of two mutants, Cm4 and Cm6 (Fig. 2A and 7A), it was still difficult to assess the inhibiting activities for viral RNA synthesis because of their low expression levels in cells. We therefore attempted to obtain cell lines expressing higher amounts of the Cm4 and Cm6 proteins. The actual levels of the mutant C proteins and of the SeV-driven luciferase expression in the newly obtained cells are shown in Fig. 7B. The relative expression levels of the four Cm4 clones were calibrated to be 431 (Cm401), 229 (Cm402), 329 (Cm403), and 184 (Cm406) and those of the four Cm6 clones to be 852 (Cm606), 629 (Cm607), 364 (Cm609), and 120 (Cm610) (Fig. 7C). The relative inhibition values of RNA synthesis calculated as above were 38 (Cm406), 47 (Cm402), 68 (Cm403), 74 (Cm401), 26 (Cm610), 44 (Cm609), 59 (Cm607), and 65 (Cm606). The plots of the relative expression levels and inhibition values of the Cm4 and Cm6 proteins clearly showed that both proteins greatly reduced the RNA synthesis-inhibiting activities compared to the C protein (Fig. 7C). The residues required for anti-RNA synthesis were thus mapped to multiple regions relevant to Cm2 (mutations at positions 77 and 80), Cm4 (positions 129 and 132), Cm5 (positions 151,152, and 153), Cm6 (position 167), and Cm8 (positions 173, 175, and 176), in contrast to the region responsible for the IFN antagonism that was mapped to a single region. Therefore, anti-RNA synthesis activity appeared to be governed by amino acids scattered across the carboxyl terminal half of the SeV C protein.


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DISCUSSION
 
The SeV C protein is a multifunctional protein, playing roles in viral RNA synthesis inhibition, IFN antagonism, apoptosis inhibition, and virus assembly (reviewed in reference 37). In previous studies (23, 24), the former two functions, viral RNA synthesis inhibition and IFN antagonism, were not segregated topologically, being mapped to the carboxyl-terminal 106 aa (positions 99 to 204) of the C protein. Here, we have generated cell lines constitutively expressing the various C protein mutants with substitution of charged amino acids for alanine residues and now show a clear dissociation of amino acid residues critically involved in RNA synthesis inhibition and IFN antagonism.

Of the point mutations investigated, only those at 151, 153, and 154 (Cm5) resulted in the loss of IFN antagonism. A similar loss of function was caused by a single point mutation (phenylalanine to serine) at position 170 (CF170S) (8, 9). However, both the Cm8 protein with nearby substitutions (173, 175, and 176) and the Cm6 protein with a substitution at an in-between position (position 156) fully retained anti-IFN capacity. Taken together, these data suggest that particular residues rather than a particular region play a critical role in IFN antagonism by the SeV C protein. In contrast, all mutants except Cm3 (substitutions at positions 114 and 115) were found to be impaired in viral RNA synthesis inhibition. Thus, RNA synthesis inhibition appeared to be governed by amino acids scattered across the primary structure of the C protein. Of those mutants displaying such an impaired phenotype, four had mutations within the 106-residue C-terminal half, whereas one (Cm2) had substitutions (at positions 77 and 78) outside the C-terminal half. The substitution at positions 77 and 78 thus appeared to critically affect the proper conformation of C protein to exhibit RNA synthesis inhibition, although the N-terminal half containing residues 77 and 78 in the natural C protein was not required for RNA synthesis inhibition (23).

How the SeV C protein inhibits homologous or closely related viral RNA synthesis has not been fully understood yet. The specific and quantity-dependent inhibition of C protein for RNA synthesis is thought to contribute terminating viral transcription and replication and promote encapsidation of virions in the late stage of viral infection. Paramyxovirus RNA synthesis is mediated by the complex comprising the L and P proteins (14). Although the detailed biochemistry of RNA synthesis yet remains to be elucidated, the L protein may act as a catalytic subunit and the P protein as a regulatory subunit containing the polymerase cofactor module and the nascent chain assembly module (reviewed in reference 33). Coauthors of the present study, Cortese-Grogan and Moyer, previously examined the binding capacity of the C mutants used here with the L protein and found that only the Cm3 retained a binding capacity as strong as that of the wild-type C, whereas all other mutants had a significantly reduced binding capacity (14). Thus, there appears to be a nice correlation between the retention or loss of the L binding capacity described above and the retention or loss of RNA synthesis inhibition found here. It therefore appears likely that RNA synthesis inhibition by SeV C protein involves at least the binding of the C protein with the L protein. The parallelism of C-L binding and RNA synthesis inhibition was, however, not as clear as seen here, when RNA synthesis was measured by using an in vitro RNA synthesis system (14); actually, the retention of the anti-RNA synthesis function of Cm3 was not very clear. The reason for this discrepancy between the in vitro RNA synthesis system and natural viral infection with a full-length genome is difficult to explain. The Cm3 protein that should be active in the context of natural infection might not act in such a way, presumably because of its accidental transinteractions with some components derived from the T7 polymerase-expressing vaccinia virus used to produce L, P, N, and C proteins for the in vitro RNA synthesis system.

Our present study further demonstrated the utility of the various C mutants to address the mechanisms of anti-IFN action exerted by the SeV C protein. Previous papers showed that the C, Y1, and Y2 proteins, all capable of blocking IFN signaling, could bind STAT1, whereas CF170S, which was incapable of blocking, failed to do so, suggesting the importance of the C-STAT1 interaction in IFN antagonism (8, 12). Our present study showed that Cm3, Cm4, Cm6, and Cm8 retained anti-IFN capacity but did not bind STAT1 stably. In addition, the formation of HMWCs possessing the C protein and STAT1 as their components was previously suggested to be relevant to blocking IFN signaling (53) but was not always seen here, with some C mutants retaining anti-IFN capacity. Thus, a C-STAT1 interaction strong enough to be coimmunoprecipitated and HMWC formation were indicated to be events nonessential for counteracting the antiviral action of IFNs.

On the other hand, when IFN antagonism was retained, both the inhibition of the phosphorylation of STAT2 (12) and increase of pY-STAT1 (27, 48) were always observed as also seen for the wild-type C protein. In contrast, when IFN antagonism was lost (Cm5), the patterns of phosphorylation (and dephosphorylation) were similar to those seen under normal IFN signaling in the parental cells. These data, taken together, suggest that the SeV C protein can induce abnormal phosphorylation and dephosphorylation of STAT proteins so that IFN signaling is blocked and that for this action a direct interaction of the C protein with STAT1 may not always be required. The importance of the inhibition of STAT2 (but not STAT1) phosphorylation for SeV C-mediated anti-IFN action was also reported recently (12). There may be an additional host factor(s) for the C protein to interact with the abnormal phosphorylation of STATs and the ultimate outcome, IFN antagonism. In searching for such host molecules, some C mutants described here will be useful; two-hybrid screening, for instance, using the wild-type C and Cm5 proteins as baits appears to be worthy of execution.


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ACKNOWLEDGMENTS
 
This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology 3, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan. Phone: 81 42 561 0711, ext. 530. Fax: 81 42 567 5631. E-mail: akato{at}nih.go.jp. Back


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Journal of Virology, July 2004, p. 7443-7454, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7443-7454.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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