Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the

The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C83S, SeV M-C106S, and SeV M-C295S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C83S and SeV M-C106S had smaller virus particles than did the wild-type SeV, whereas SeV M-C295S had larger and heterogeneously sized particles. Furthermore, SeV M-C106S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle. Sendai virus (SeV), an enveloped virus with a single-stranded negative-sense RNA genome of 15,384 bases, belongs to the genus Respirovirus of the family Paramyxoviridae. The virus particle displays spherical morphology of relatively uniform size with a diameter of about 200 nm. The envelope comprises a lipid bilayer derived from the host plasma membrane and two inserted viral glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) proteins. Lining beneath the envelope are the matrix or membrane (M) proteins. The nucleocapsid represents the internal structure, which comprises the genome RNA complexed with the nucleocapsid (N) protein and polymerase consisting of the L (large) protein and the P (phospho) protein (11). There is increasing evidence suggesting that the M protein plays a key role in the assembly of paramyxoviruses and related RNA viruses. The M protein has been suggested to be essential to cross-link the external envelope proteins and the internal nucleocapsid. It also promotes the condensation of viral glycoproteins into a patch in the plasma membrane, an immediate precursor of the envelope (16, 28). The M proteins, associating with the nucleocapsid, may then provide forces from the inside for the membrane patch to bud (3, 7, 15). Bending of membranes may also be facilitated from the outside by glycoproteins (17, 18). We previously showed that the SeV M protein expressed from plasmid was released into the culture supernatant, as was seen in the case of the M protein of vesicular stomatitis virus (12, 22). Recently, the F protein as well as the M protein was shown to cause the budding of vesicles from cells (25). The SeV M protein as well as the F protein therefore has an intrinsic nature to be a driving force of virus budding. The SeV M protein is 348 amino acids in length and contains five cysteine residues. Cysteines can form intrachain and interchain disulfide bonds and thereby contribute to the folding of polypeptides as well as homologous and heterologous proteinprotein covalent interactions. Cysteines in various enzymes function as an active center, and those in various proteins sometimes form zinc finger motifs to bind metabolically important zinc ions (5, 13). The last function has recently been exemplified for a nonstructural protein of SeV, the V protein (8). Little is known, however, about the cysteine residues of the SeV M protein. It is unlikely that the cysteines in the M protein are intracellularly oxidized to form disulfide bonds because the M protein is localized in the cytosol, the reducing milieu. However, it is not known whether the cysteines of the M protein form disulfide bonds in virus particles in an oxidizing extracellular environment. Nevertheless, the fact that cysteine residues are well conserved in a wide variety of paramyxovirus M proteins suggests that the functions of the M proteins are important. We therefore focused on cysteine residues at the targets of site-directed mutagenesis and investigated the actual contribution of the M protein to SeV assembly.