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. 2004 Jul;78(14):7443-54.
doi: 10.1128/JVI.78.14.7443-7454.2004.

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

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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 et al. J Virol. .
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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.

Figures

FIG. 1.
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-β. (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-β 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.
FIG. 2.
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-β. (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-β by using VSV as a challenge virus as described in the legend of Fig. 1B. (C) Antiviral activities of IFN-β (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.
FIG. 3.
FIG. 3.
Expression of IFN-responsible genes in the parental and C-expressing HeLa cells. Induction of ISG products, STAT2, STAT1α/β, 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-β 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.
FIG. 4.
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.
FIG. 5.
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-β/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).
FIG. 6.
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.
FIG. 7.
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.

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