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-α-mediated anti-viral state. Infection by the virus (Cm2′) containing mutations at K77 and D80 induced significant IFN-β 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-β 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.
Highlights
Sendai virus (SeV; mouse parainfluenza virus type 1), a prototype of the family Paramyxoviridae of the order Mononegavirales which includes some of the most important and ubiquitous disease-causing viruses of humans and animals, such as measles virus, parainfluenza viruses, mumps virus, Nipah virus, Hendra virus, human metapneumovirus, Newcastle disease virus, canine distemper virus, and rinderpest virus, contains a nonsegmented, negative-stranded RNA genome with length of 15,384 nucleotides [1]
It has been reported that clusters of charged amino acids conserved within C proteins among SeV strains are important for the inhibition of (+)-sense viral RNA synthesis [17], and that some of the charged amino acids play an important role in the inhibitory action against IFN signaling [11]
As for the other C mutant viruses, the (+)/(2) ratio in virions was virtually identical to that detected in the WT virion except in the Cm29 virion, in which it was 4.5-fold higher than that in the WT virion (Fig. 3). These results indicate that the conserved charged amino acids in C proteins do not play critical roles in the regulation of viral RNA synthesis at the level observed in the C-knock out viruses
Summary
Sendai virus (SeV; mouse parainfluenza virus type 1), a prototype of the family Paramyxoviridae of the order Mononegavirales which includes some of the most important and ubiquitous disease-causing viruses of humans and animals, such as measles virus, parainfluenza viruses, mumps virus, Nipah virus, Hendra virus, human metapneumovirus, Newcastle disease virus, canine distemper virus, and rinderpest virus, contains a nonsegmented, negative-stranded RNA genome with length of 15,384 nucleotides [1]. It has been reported that the substitution of three charged amino acid residues at positions 151, 153, and 154 within C protein conserved among SeV strains dramatically reduced the anti-IFN ability without any alteration of the other functions of C protein in experiments with C protein expressed from cDNA as well as infections of SeV recombinants [10,11] These substitutions led to an attenuation of the SeV recombinant in mice in a STAT1-dependent manner, but not to reduced viral growth in cell cultures [10]
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