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

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-β, 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-α or -β and IFN-γ (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-α/β system.