The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis

Abstract

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.

FIG. 1.

Native and truncated C proteins expressed from the reading frame overlapping the P frame on SeV P mRNA. (A) The C proteins (open bars) cloned into the expression vector are shown on the P mRNA. The amino-terminal portion of the P protein is shown as the filled bar. The numbers represent the nucleotide positions from the start site of P mRNA. The amino acid length of each C protein is shown in the box at right. (B) Immunoprecipitates of HeLa cell lines expressing native or respective truncated C proteins are shown. The positions of molecular mass markers (in kilodaltons) are shown at left.

FIG. 2.

Expression of IFN-responsible STAT1, STAT2, and PKR proteins in the parental and C-expressing HeLa cells. The amounts of STAT1α/β, STAT2, and PKR proteins are shown in each immunoblot. The densities of the bands taken in each lane were calibrated as described in Materials and Methods. IFN-β was applied for the indicated number of hours. The stimulation ratios between incubations in the absence and the presence of IFN-β are shown in the boxes. The assays for parental, C+, Y1+, and Y2+ HeLa cells (A) and for cells expressing the respective truncated C proteins (B) are indicated.

FIG. 3.

Formation of ISGF3 complex in the IFN-stimulated cells. The formation of ISGF3 complex was assayed by the mobility of the DNA fragment (IRE). The positions of the shifted IRE fragments bound with the ISGF3 complex are indicated by arrows. The nonspecific bands caused by incubation with cell extracts are indicated by asterisks. The assays for parental, C+, and Y1+ HeLa cells (A), for Y2+, Y2.5+, and Y3+ HeLa cells (B), and for Y4+, Y8R+, and Y7R+ HeLa cells (C) are shown. Labeled IRE, unbound free IRE probe.

FIG. 4.

IFN-induced antiviral states of the parental and C-expressing HeLa cells. The cells were incubated for 24 h with various amounts of IFN-β (A) or IFN-γ (B) as indicated. They were then challenged with VSV or mock infected (Mock). Cells that survived the challenge infection and attached to the plates were fixed and stained.

FIG. 5.

Reporter gene expression from the SeV minigenome or the rSeV genome in the parental and C-expressing HeLa cells. (A) The firefly and Renilla luciferase activities expressed from the minigenome and the control plasmid, respectively, were calibrated in the various cells. Relative luciferase activities are expressed as mean values with error bars at left, and percentages are given at right. (B) The luciferase activities per microgram of protein, with error bars, are shown at left, and percentages are shown at right. Filled bars and open bars indicate the activities examined under the conditions with and without proteasome inhibitor (MG-132), respectively.

FIG. 6.

Virus growth in the parental and C-expressing HeLa cells. (A) The SeV titers in the culture supernatants at several incubation times are plotted for the parental and C-expressing HeLa cells. (B) The SeV titers at 18 h (filled bars) and 24 h (open bars) are expressed as percentages of the respective titers of the parent cells. (C) hPIV1 (left) and hPIV3 (right) were inoculated into the parental (open circles) and Y2+ (filled circles) HeLa cells. The virus titers at several incubation times are shown.