Saiji Yoshii

Membrane (M) protein of HVJ (Sendai virus): Its role in virus assembly

Abstract The role in virus assembly of membrane or matrix (M) protein of HVJ (Sendai virus) was investigated. When virions were disrupted with alkali Tween 20, nucleocapsids with a sheathlike cover on the surface and a width of approx 33 nm were often obtained, and this sheathlike structure was composed of M protein. Selective aggregation of viral components in vitro was examined. Each viral component was isolated separately from virions by using detergents, and they were mixed with each other in various combinations. The structures which reaggregated were examined for their protein composition, and the results indicate that the nucleocapsid protein does not combine with viral glycoproteins to form a coaggregate unless M protein is present, which suggests that the M protein in vivo may be a mediator for the association of nucleocapsid with the modified areas of plasma membrane which will become viral envelope.

Growth of Newcastle disease virus in a HVJ carrier culture of HeLa cells

Abstract Growth of Newcastle disease virus (NDV) in a HVJ carrier culture of HeLa cells (HeLaHVJ) was investigated. Although most of the cells of the carrier culture contained HVJ antigen, its presence did not interfere with the growth of superinfecting NDV. When HeLaHVJ cells were infected with NDV at a low multiplicity of infection, the yield of progeny virus was higher, and the cytopathic changes were more extensive than those in normal HeLa cells. Plaques produced by NDV on HeLavHVJ cell monolayers were clearer and larger than those on HeLa cells. HeLaHVJ cells could be distinguished from normal HeLa cells both with respect to production of interferon and sensitivity to its action. Production of interferon which normally appeared in HeLa cell culture infected with NDV could not be demonstrated in HeLaHVJ cell culture. The cells of HeLaHVJ were less susceptible to the antiviral action of interferon than normal HeLa cells.

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Twenty‐three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15‐fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat‐stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.

Analysis of the inhibitory effect of canavanine on the replication of influenza RI/5+ virus. I. Inhibition of assembly of RNP.

Abstract When influenza A2/RI/5+ virus-infected 1-5C-4 cells were incubated in medium containing 2 μg/ml of canavanine (an arginine analog) from 4 hr after infection, virus growth was completely inhibited. The mechanisms of inhibition by canavanine were investigated by immunofluorescent staining or isotope labeling of cells. The results indicated that in canavanine-treated cells all known viral proteins were synthesized, but most of the nucleocapsid protein (NP) and nonstructural protein (NS) was present in the nucleus, in contrast to control cells in which they were distributed throughout the whole cells. Further, formation of viral ribonucleoprotein (RNP) was inhibited and most NP was present in a nonassembled, soluble form. This action of canavanine was reversible. When arginine was added to canavanine-treated cells, viral RNP soon became detectable in the cytoplasm, and this was followed by the production of infectious virus. However, simultaneous addition of cycloheximide with arginine did not restore the formation of RNP or virus production. Based upon these findings, it is suggested that the inhibitory effect of canavanine on the replication of influenza A2/RI/5+ virus is inhibition of assembly of viral RNP.

Mechanism of interference between influenza A/WSN and B/Kanagawa viruses.

Simultaneous infection of MDCK cells with influenza viruses A/WSN and B/Kanagawa resulted in mutual interference with virus protein synthesis and in significant suppression of A/WSN growth. When infection by one virus preceded the other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level of transcription. Interference by the pre-infecting virus was strongly dependent on the expression of the viral genome but not on haemagglutinin activity. When the replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by actinomycin D. These findings suggest that intertypic interference occurs at the level of primary transcription. This concept was supported further by the observation that a ts mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with superinfection by B/Kanagawa at the permissive temperature.

Modification of normal cell surface by smooth membrane preparations from BHK-21 cells infected with Newcastle disease virus

Smooth membrane fractions were prepared from the cytoplasmic extract of BHK-21 cells infected with Newcastle disease virus (NDV). These membranes exhibited high hemagglutinating, neuraminidase, and hemolytic activity but little infectivity, suggesting that they might be precursors for viral envelope. When such membranes were adsorbed to the monolayers of uninfected BHK-21 cells at 4‡C and then incubated at elevated temperature for a couple of hours, the cells became highly hemadsorptive even in the presence of cycloheximide. This phenomenon occurred between 15‡C and 25‡C, and was maximal at 31‡C, where approximately 4 times more erythrocytes were adsorbed than to the cells incubated at 4‡C. Immunofluorescent staining suggested that diffusion of viral antigens might occurred rapidly over the entire surface of the cells.Cell fractions containing virions induced hemadsorption in uninfected cells, too. However, induction occurred now at 31‡C and was maximal at 37‡C, and erythrocytes appeared to be adsorbed not to the entire surface of the monolayer but restricted areas of the cells. The diffusion of viral antigens on the cell surface was not so significant under these conditions.On the basis of these findings the possible role of the membranes of the smooth endoplasmic reticulum in virus replication is discussed.

Plaque variants of Sindbis virus

Large plaque (G) and small plaque (8) variants were cloned from stock culture of Sindbis virus. Addition of protamine to agar overlay was shown to have little or no effect on the size of plaques produced by G variant, whereas S virus plaque size was significantly increased. This finding suggests that the S variant is more susceptible than the G variant to the inhibitory action of agar factor. The G and S variants differed markedly in their sensitivity to the antiviral action of interferon. Evidence was obtained that the S variant is more susceptible to the action of interferon. The difference of G and S variants in susceptibility to the action of interferon and agar factor may account for the difference of their plaque size. Growth and cytopathic effect of the S variant in a HVJ carrier HeLa cell culture (HeLaHVJ) was greatly enhanced in contrast to those in normal HeLa cells. Enhancement of growth of wild-type virus in HeLaHVJ cells was not so significant as in the case of the S variant. These observations were discussed in relations to the properties of the S variant and the cells of HeLaHVJ.

Intracellular Development of Membrane Protein of Influenza Virus

The intracellular development of membrane protein (MP) of influenza A virus was investigated by immunofluorescent staining. Monospecific antiserum was prepared by immunizing rabbits with MP eluted from SDS‐polyacrylamide gels of SDS‐disrupted NWS virions. In the productive infection in clone 1‐5C‐4 cells, MP antigen was first detected over the whole cell at 4 hr after infection, concomitantly with the appearance of hemagglutinin (HA) antigen in the cytoplasm, and bright nuclear fluorescence was then observed. Nucleoprotein (NP) antigen was detected in the nucleus prior to the appearance of fluorescence of MP antigen and thereafter the cytoplasmic fluorescence developed. Late in infection, all of these three antigens were observed predominantly in the cytoplasm with stronger fluorescence at the cell surface. Essentially similar findings were obtained in the abortive infections in L cells and BHK cells.

Surface antigens on HeLa cells persistently infected with HVJ (Sendai virus).

SummarySurface antigens of HeLaHVJ cells, a cell line persistently infected with HVJ, were studied by fluorescent antibody staining. After absorption with concentrated HVJ virions and HeLa cells, anti-HeLaHVJ antiserum was able to demonstrate specific surface fluorescence on HeLaHVJ cells, while this serum no longer reacted with original HeLa cells nor with HVJ virions. During cytolytic infection of HeLa cells with HVJ, this specific surface antigen appeared at an early stage of infection prior to the appearance of newly synthesized HVJ viral antigens and moreover appeared in spite of the inhibition of viral protein synthesis. This antigen was detected neither on HeLa cells infected with other myxoviruses except HVJ nor on various other kinds of cells infected with HVJ. The specific surface antigen was still found on the HeLaHVJ cell surface after incubation at 38° C for two days, while HVJ structural antigens on the cell surface no longer could be detected. Mild short-term treatment of HeLa cells with trypsin, neuraminidase fromvibrio cholerae, phospholipase-C and hyaluronidase failed to expose specific antigen. The antigen was distinguishable from the Forssman and human blood type antigens. The mechanism of appearance of a new antigen on the surface of HeLaHVJ cells remains unclear.

Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature‐sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5‐fluorouracil and classified them into seven recombination groups by pair‐wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis‐positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10–2 and belonged to recombination group VII.