Conserved charged amino acids within Sendai virus C protein play multiple roles in the evasion of innate immune responses

Abstract

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C'/C(-), 4C(-), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-alpha-mediated anti-viral state. Infection by the virus (Cm2') containing mutations at K77 and D80 induced significant IFN-beta production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2' virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-beta and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.

Figure 1. SeV recombinants used and their growth kinetics.

(A) Schematic representation of the SeV genome highlighting the start region of the P gene including the C ORF. The amino acid changes for the various mutants are indicated. (B) Graphs of one-step growth kinetics of the viruses in LLC-MK₂ cells. Each titer represents the average for at least three independent experiments.

Figure 2. Protein profiles for WT and recombinant viruses.

(A) SDS-PAGE analysis of virion proteins released and accumulated for 48 h p.i. in the culture medium of LLC-MK₂ cells infected with the indicated viruses and Western blots for N, P, and C protein expression in the infected cells. (B) The amounts of N protein in virions (vN) and in the infected cells (cN) in Fig. 2A were quantitated and the ratio of vN to cN is shown as a bar graph. The ratio in SeV-WT was set to 1. Bars represent the average for three independent experiments.

Figure 3. Viral genomic and antigenome-sense RNA incorporated in virions.

Ratios of (+)- to (−)-sense RNAs in the indicated virions, which were detected by two-step qRT-PCR, are shown as a bar graph. The ratio of the WT-sample was set to 1. Bars represent the average for three independent experiments.

Figure 4. Cytopathogenicity of the WT and recombinant viruses.

(A) Microscopic analysis of virus-induced CPE. LLC-MK₂ cells were mock-infected or infected with the indicated viruses at an MOI of 5 and cells were observed at 24 and 48 h p.i. (B) Cytotoxicity assay using LLC-MK₂ cells. LLC-MK₂ cells were infected with the indicated viruses at an MOI of 5. The lactate dehydrogenase (LDH) activity released from the damaged cells for 48 h after infection was measured as described in the Materials and Methods section. The value of the WT-sample was set to 1.

Figure 5. Ability of the WT and recombinant viruses to rescue VSV from the anti-viral action of IFN-α.

HeLa cells were mock-infected or infected with the indicated viruses. At 6 h p.i., the media were replaced with fresh media containing IFN-α (1,000 IU/ml) or no IFN-α. After an additional 6-h incubation, cells were superinfected with rVSV-GFP. After further incubation for 6 h, GFP expression derived from rVSV-GFP replication was observed under a fluorescent microscope. (B) Cell lysates were analyzed by Western blotting to detect expression of rVSV-GFP-derived GFP, SeV P, and C proteins. (C) The amounts of GFP in the cell lysates of Fig. 5B were quantitated and shown as a bar graph. The value of the mock-infected, IFN-α-non-treated sample was set to 1. Bars represent the average for three independent experiments.

Figure 6. IFN-β production in the HeLa cells mock-infected and infected with the indicated viruses.

The relative amounts of IFN-β mRNA detected in the cells infected with the indicated viruses at 48 h p.i. revealed by one-step qRT-PCR are shown as a bar graph. The amount of the mRNA detected in the mock-infected sample was set to 1.

Figure 7. Immunofluorescent staining of the SeV-infected cells with an anti-dsRNA mAb.

HeLa cells were mock-infected or infected with the indicated viruses. At 36 h p.i., cells were fixed, permeabilized, and then stained with an anti-dsRNA mAb J2, anti-SeV pAb, and DAPI as described in the Materials and Methods section. Cells were observed under a Zeiss LSM5 confocal microscope.

Figure 8. Subcellular distribution of cellular IRF-3 in the SeV-infected cells after induction by poly(I:C).

HeLa cells were mock-infected or infected with the indicated viruses. At 12 h p.i., cells were transfected with 5 µg of poly(I:C) and incubated for another 6 h. Cells were fixed, permeabilized, and then stained with anti-IRF-3 pAb and anti-SeV N mAb as primary antibodies. Cells were observed under a Zeiss LSM5 confocal microscope.