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, 74 (12), 5619-28

Versatility of the Accessory C Proteins of Sendai Virus: Contribution to Virus Assembly as an Additional Role

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Versatility of the Accessory C Proteins of Sendai Virus: Contribution to Virus Assembly as an Additional Role

M K Hasan et al. J Virol.

Abstract

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.

Figures

FIG. 1
FIG. 1
(A) Replication of WT and 4C(−) viruses in CV1 cells. (B) CIU-to-HAU ratios of the yields. (C) Immunoelectron micrographs of CV1-grown WT and 4C(−) virions. Bar = 100 nm.
FIG. 2
FIG. 2
Western blotting (A) and Northern blotting (B) of CV1 cells infected with WT and 4C(−) viruses and RT-PCR-based analysis of genome replication of the infected cells (C).
FIG. 3
FIG. 3
Synthesis and incorporation into virions of viral proteins. (A) CV1 cells infected with WT and 4C(−) viruses were pulse labeled with Tran35S protein labeling mix for 1 h at various hours p.i. indicated on the top of each gel lane, and the cell lysates were processed for immunoprecipitation with anti-SeV polyclonal antibody and SDS-PAGE. (B) At 20 h p.i., the labeled cells were chased for the periods indicated and the virions isolated from culture supernatants were analyzed by SDS-PAGE. The dots on the left of WT Chase lane 1 indicate the positions of HN, F0, and M proteins. (C) 35S-labeled WT and 4C(−) viruses were isolated from the supernatants of infected CV1 cells by adsorption onto and elution from chicken RBC and centrifuged through a 20 to 60% linear gradient of sucrose. Fractionated materials were analyzed by SDS-PAGE (t, top; b, bottom).
FIG. 4
FIG. 4
Intracellular distribution of SeV proteins. CV1 cells infected with WT and 4C(−) viruses were fixed, permeabilized, and stained with anti-P, anti-L, and anti-HN antibodies separately at 20 h p.i. (for P and HN) or 40 h p.i. (for L).
FIG. 5
FIG. 5
Double antibody staining of WT-infected cells with anti-C antibody (green) and anti-P, anti-M, or anti-HN antibody (red). Cells were fixed at 20 h p.i. Merged images in each combination are also shown.
FIG. 6
FIG. 6
Response of CV1 and Vero cells to IFN-α/β and incorporation of viral proteins into virions in Vero cells. (A) CV1 and Vero cells transfected with pISRE-luci were incubated in the presence (filled bars) or absence (open bars) of human IFN-α/β for the different hours indicated and assayed for luciferase activities. (B) Incorporation of viral proteins synthesized in infected Vero cells into the virion fractions of the supernatants was analyzed by pulse-chase labeling exactly as was done in Fig. 3. The dots on the left of WT chase lane 1 indicate the positions of HN, F0, and M proteins.

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