Showing papers by "Cheng Huang published in 2002"

Involvement of the Leader Sequence in Sendai Virus Pathogenesis Revealed by Recovery of a Pathogenic Field Isolate from cDNA

Abstract: We previously demonstrated that a systematic passage of a pathogenic field isolate of Sendai virus (SeV), the Hamamatsu strain, in embryonated eggs caused attenuation of virulence to mice, and we isolated viral clones of distinct virulence (K. Kiyotani et al. Arch. Virol. 146:893-908, 2001). One of the clones, E15cl2, which was obtained from the virus at the 15th egg passage of E0, the parental Hamamatsu clone for egg passage, had 165-fold-attenuated virulence to mice and possessed only four mutations in the entire 15,384-base genome: in an antigenomic sense, U to A at position 20 (U20A) and U to A at position 24 (U24A) in the leader sequence, the promoter for transcription and replication, and A to G at position 9346 (silent) and A to U at position 12174 (Ser to Cys) in the L gene. To examine the possibility that leader mutations affect virus pathogenesis, we recovered live viruses from cDNA derived from the Hamamatsu strain. A mutant virus possessing either a mutation of U20A or U24A in the leader sequence showed a slightly lower pathogenicity than that of the parental virus, whereas a double mutant virus possessing both of the mutations showed 25-fold-attenuated virulence, accompanying a significantly lower virus replication in the mouse lung. Replications of the leader mutant viruses were also impaired in a primary culture of mouse pulmonary epithelial cells but not in chicken embryo fibroblasts. These findings suggest that leader mutations of SeV affect virus pathogenesis by altering virus replication in a host-dependent manner.

Identification of mutations associated with attenuation of virulence of a field Sendai virus isolate by egg passage.

Abstract: We have reported that attenuation of the virulence of a field Sendai virus (SeV) isolated by egg passage is associated with an impediment of viral genome replication in mouse respiratory cells (Kiyotani et al., Arch Virol 146, 893–908, 2001). To determine the molecular basis for the attenuation, we sequenced entire genomes of representative SeV clones isolated during egg passages and compared those with that of the parental SeV clone E0. E15cl2, a 165-fold attenuated clone in 50% mouse lethal dose (MLD50) isolated at the 15th egg passage, possessed only four mutations in the entire genome: U to A at position 20 (U20A) and U24A in the leader promoter region and A9362G and A12174U in the L gene from the 5′-end of antigenome. The former mutation in the L gene was silent and the latter changed deduced amino acid Ser at position 1207 to Cys (Ser1207Cys) in the L protein, a catalytic subunit of viral polymerase. E30cl2, a further 6-fold attenuated clone isolated at the 30th egg passage, had an additional four mutations: A8074G (Glu461Gly) and A8077G (Asp462Gly) in the hemagglutinin-neuraminidase (HN) gene and A13598C (silent) and G13927A (Ser1791Asn) in the L gene. On the other hand, a virulent revertant clone, E30M15cl5, which was obtained by 15 mouse passages of E30cl2 and had 250-fold mouse virulence compared to E30cl2, possessed eight mutaions: A24U in the leader, C1325U (silent) in the nucleocapsid gene, G8074A (Gly461Glu) in the HN gene, G10433U (Lys626Asn), C13598A (silent), A13927G (Asn1791Ser), C14626U (Thr2024Ile) and A15272C in the L gene. Among these, the mutations in the leader and the HN gene and two of the mutations in the L gene (C13598A and A13927G) were true reversions to E0. The significance of the mutations detected in the leader as well as in the L and HN genes was discussed in the context of attenuation of SeV pathogenicity by egg passage.

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

Abstract: 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 protein-protein 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.

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

Abstract: 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.