Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.

No abstract

Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C, C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions Sendai virus (SeV) is an enveloped virus with a linear, nonsegmented, negative-sense RNA genome of 15,384 nucleotides and belongs to the genus Respirovirus of the subfamily Paramyxovirinae. SeV contains six genes in the order 3Ј-(leader)-N-P-M-F-HN-L-(trailer)-5Ј on the genome. The monocistronic mRNAs are transcribed by the viral RNA polymerase composed of L and P proteins (16). However, the P gene is exceptional in that it gives rise to multiple protein species by a process known as RNA editing and by the use of an overlapping open reading frame (ORF). The P protein is translated from the unedited mRNA, which is the exact copy of the P gene, while the V protein is translated from the edited mRNA, in which one nontemplated G residue is cotranscriptionally inserted to the editing position. The P and V proteins, therefore, have a common N terminus but have different C termini because the reading frame for V changes by ϩ1 relative to the P frame (for a review, see references 33 and 38). The V protein is encoded by almost all viruses that are members of the subfamily Paramyxovirinae. The unique C-terminal region of V contains seven cysteine residues highly conserved among paramyxoviruses, forms zinc finger-like motifs, and, indeed, binds Zn 2ϩ (7,20,35,44,51). The C protein is translated from the Ϫ1 reading frame relative to the frame common to P and V. The SeV P and V proteins are initiated at the AUG codon at position 104, whereas the SeV C reading frame produces a nested set of CЈ, C, Y1, and Y2 proteins initiating, respectively, at a non-AUG (ACG) codon at position 81 and at AUG codons at positions 114, 183, and 201 (4, 15, 45). The CЈ, C, Y1, and Y2 proteins are terminated at the same position 726 and are collectively called C proteins. Among them, the C protein is the major species expressed in infected cells, at a molar ratio severalfold higher than that of the other three proteins (32). The C proteins are expressed by the viruses belonging to three genera, Respirovirus, Morbillivirus, and Henipahvirus, and by Tupaia paramyxovirus-like viruses, but they are not expressed by the viruses belonging to two genera, Rubulavirus and Avulavirus of Paramyxovirinae (reviewed in reference 37). The amino acid sequence of the C proteins is well conserved within each genus

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)‐infected LLC‐MK2 cells with 50 μM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose‐dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV‐like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC‐MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell‐specific and partly virus‐specific.

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted.