Y2, the smallest of the Sendai virus C proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral RNA synthesis

Abstract

An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C', C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-alpha/beta) and IFN-gamma and to inhibit viral transcription. The assay protocols included monitoring of IFN-alpha/beta-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-alpha/beta and IFN-gamma and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(-) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

FIG. 1

Expression of P, V, and C proteins from the SeV P gene and positions of the primer-probe sets used in this study. The locations on the genome of the primer and probe pairs used to detect genome RNA and N mRNA are shown. The nucleotide numbering is from the 3′ end of the viral genome. For coding strategies of the various proteins, see the text. The numbers represent the nucleotide positions from the start site of the P mRNA.

FIG. 2

Expression of C, Y1, or Y2 protein in stable cell lines. Immunoblotting (A) and immunofluorescent staining (B) of C⁺, Y1⁺, and Y2⁺ cells are shown. All four C proteins expressed in SeV-infected HeLa cells were used as markers (SeV). Relative expression levels of C, Y1, and Y2 are shown (A, bottom). None indicates the parental HeLa cell line in this and subsequent figures.

FIG. 3

Relative luciferase activities and expression of STAT1 and STAT2 in parental and C-expressing HeLa cells. Reporter luciferase plasmid pISRE-luci driven by the ISRE and control luciferase plasmid pRL-TK driven by the tk promoter were transfected (for details, see Materials and Methods). Relative luciferase activities were measured at 0, 2, 4, and 6 h of incubation with (solid bars) or without (open bars) INF-β. The amounts of STAT1α/β and STAT2 proteins in the presence of IFN-β (A) or IFN-γ (B) at the times indicated are shown in each immunoblot.

FIG. 4

IFN-induced antiviral states of parental and C-expressing HeLa cells. Cells were incubated for 24 h with various concentrations of IFN-β (A and B) or IFN-γ (C) as indicated. Subsequently, they were challenged with VSV or mock infected (Mock). Cells which survived the challenge infection and were attaching to the plates were fixed and stained (A and C). Viral multiplication was detected by immunoblotting with anti-VSV serum (B). Viral proteins are indicated on the left.

FIG. 5

SeV multiplication in parental and C-expressing HeLa cells. Cells were infected with 4C(−) (A) or Wt (B) SeV. The virus titers in the culture supernatants were determined at the times indicated.

FIG. 6

Reporter gene expression from the SeV minigenome in parental and C-expressing HeLa cells. In addition to C⁺, Y1⁺, and Y2⁺ cells, three other Y2-expressing lines, Y2a⁺, Y2b⁺, and Y2c⁺, were used. (A) Expression of the respective C proteins in these cells, determined by immunoblotting, along with their relative expression levels (top). Relative luciferase activities expressed from the minigenome in the various cells are shown along with the mean value (black bars) for two measurements (bottom). (B) Relationship between the luciferase activity from the minigenome and the intracellular expression level of each C protein.

FIG. 7

Quantification of SeV primary transcripts in parental and C-expressing HeLa cells. (A) Cells were infected with Wt SeV at an MOI of 20 per cell and incubated for 5 (gray bars) or 10 (black bars) h in the presence of cycloheximide (100 μg/ml), and total RNAs were extracted. The copy numbers of N mRNA (top) and genome RNA (bottom) were then quantified by TaqMan PCR with specific primers and probes. Relative copy numbers measured at 0 h p.i. are shown above the black bars. (B) Relationship between the relative copy number of N mRNA and the relative levels of intracellular C proteins.