Morio Homma

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells

A trypsin‐like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC‐MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin‐like protease which activates the inactive form of Sendai virus.

The synthesis of polypeptides in influenza C virus-infected cells.

The synthesis of virus-specific polypeptides was analyzed in MDCK cells infected with the JJ/50 strain of influenza C virus. In addition to three major structural proteins gp88, NP, and M, the synthesis of five polypeptides with molecular weights of 29,500 (C1), 27,500 (C2), 24,000 (C3), 19,000 (C4), and 14,000 (C5) was found in infected cells. None of these polypeptides were detected either in virions or in immunoprecipitates obtained after treatment of infected cell lysates with antiviral serum, suggesting that they are not viral structural proteins. Polypeptides C1-C5 were found to be synthesized in MDCK cells infected with different influenza C virus strains as well as in different host cell types infected with C/JJ/50. Further, it was observed that cellular protein synthesis was greatly reduced under hypertonic conditions, whereas the synthesis of C1-C5 was relatively unaffected. These results suggest that polypeptides C1-C5 are virus coded rather than host cell coded. Peptide mapping studies showed that each of polypeptides C3, C4, and C5 had a peptide composition similar to the M protein. The amount of C2 synthesized in infected cells was insufficient for mapping. This polypeptide was, however, found to rapidly disappear in pulse-chase experiments, suggesting that C2 is probably not unique but biosynthetically related to one of the other proteins. In contrast to these polypeptides, polypeptide C1 showed a map which is largely different from any major structural polypeptide. It therefore appears likely that C1 is a nonstructural protein of influenza C virus similar to the NS1 protein of influenza A and B viruses.

Age distribution of the antibody to type C influenza virus.

Type C influenza virus has been considered an etiologic agent of mild upper respiratory illness of human beings (6, 9, II, 15). Sera of most healthy adults have been shown to contain hemagglutination-inhibiting (HI) antibody to the virus at a significant level (I, 5, 10, II, 13). In contrast, the sera of children less than I year of age have not been shown to contain the antibody although antibody-positive sera increase with age among young children, aged I to 7 years (8). These facts suggest that the virus is widespread and that the majority of children are infected with the virus by the age of 7 years. However, outbreaks of type C influenza have seldom been encountered and their confirmation by either virological or serological means was made only retrospectively (5-7). This may be attributable to its poor manifestation of clinical signs and to the difficulty of isolating the virus from the patients. Epidemiological information on type C influenza has also been very limited as compared with that of types A and B influenza and no information is available about periodicity of the outbreaks, mode of transmission and maintenance of the virus in nature. To approach these problems, we attempted first to determine the age distribution of antibody to the virus in the residents of the same community in two surveys with a three-year interval. This kind of study has not been made previously in Japan and the results can be directly compared with those reported in countries outside Japan. Special efforts were also made to demonstrate maternal antibody in infants less than I year of age, because we have been puzzled by our own serological observation that children less than I year old seemed to be highly protected from the infection. A total of 186 human sera were collected from healthy residents in the age range of 0-80 years in Yamagata Prefecture, Japan, during the period of September to December 1976 and 434 sera were obtained from different individuals in the same community during the period of August to December 1979. Antibody levels were determined by measuring the hemagglutination-inhibition (HI) titer of the sera and the titers were expressed as the reciprocal of the highest serum dilution that inhibited hemagglutination. HI titration was conducted in microtiter plates, with 0.5% chicken erythrocytes and with phosphate buffered saline (PBS, 0.01 M

Effects of various proteases on the glycoprotein composition and the infectivity of influenza C virus

SummaryTrypsin and elastase activated the infectivity of influenza C virus by cleaving a precursor glycoprotein gp88 into subunits gp65 and gp30, whereas chymotrypsin and thermolysin did not.

Evidence of proteolytic activation of Sendai virus in mouse lung

SummaryA device was made to analyze the pneumotropism of Sendai virus in mouse. Minced lung blocks were prepared from the mouse intranasally infected with Sendai virus for 2 hours and cultured in a CO2 incubator. This culture system provided a suitablein vitro model of Sendai virus infection in mice in terms of the distribution of the viral antigens and histopathological findings. The progeny virus recovered from the lung culture was already activated and was accompanied by the cleavage of F glycoprotein into F1 and F2. This fact demonstrates that the activating mechanism is preversed in the lung culture as foundin vivo infection of mouse lung. The viral activation and the cleavage of F glycoprotein were simultaneously inhibited by tosyllysylchloromethylketone, leupeptin, soybean trypsin inhibitor and antipain, but not by tosylamidophenylethylchloromethylketone, chymostatin, pepstatin, iodoacetamide, phenylmethylsulfonylfluoride and p-chloromercuribenzoate. These results show that the activating enzyme of Sendai virus found in the lung culture was similar to trypsin. The existence of the activating enzyme may support the replication of Sendai virus in mouse lung in multiple-step and also result in the lung pathology.

Effect of tunicamycin on the replication of sendai virus

Abstract The effect of tunicamycin (TM) on the synthesis, glycosylation, and maturation of Sendai virus glycoproteins was investigated. Incorporation of [3H]glucosamine into HN and F glycoproteins was completely inhibited by TM at a concentration of 0.5 μg/ml. Treatment of infected cells with TM under these conditions caused a 106-fold reduction in the production of infectious progeny virus. Synthesis of nonglycosylated viral proteins, P, NP, and M was clearly detected in TM-treated cells while synthesis of glycoproteins HN and F was not. However, two new polypeptides with molecular weights of 63,000 (T,) and 55,000 (T2) were synthesized in the presence of TM. Tryptic peptide analysis revealed that the T1 and T2 polypeptides are the nonglycosylated forms of the HN and F proteins, respectively. When microsome vesicles isolated from TM-treated cells were treated with trypsin, T1 and T2 were found to be protected from proteolysis, which suggests that after their synthesis the HN and F proteins are properly inserted into the endoplasmic reticulum bilayer even in the absence of glycosylation. In addition, the nonglycosylated forms of glycoproteins were found in pulse-chase experiments to normally migrate from rough to smooth cytoplasmic membranes. However, they could not be detected on the surface of infected cells by direct immunofluorescent staining, suggesting that the HN and F proteins can not be integrated into plasma membranes in the absence of glycosylation. Further, electron microscopic observation showed that there were no budding particles on the surfaces of TM-treated cells.

Genetic variation among human strains of influenza C virus isolated in Japan

The RNA genomes of sixteen human strains of influenza C virus isolated in Japan between 1964 and 1983 were compared by SDS-polyacrylamide gel electrophoresis and oligonucleotide fingerprinting. A high degree of genetic variation was observed among the strains analysed. However, there were some strains with the genomes closely related to one another, and they could be divided into two groups. The first group consists of C/Shizuoka/79, C/Kanagawa/1/81 and four strains of C/Yamagata/81. The 1981 strains of this group were all isolated in March of the year. The second one consists of C/Kyoto/41/82, C/Nara/82 and C/Hyogo/1/83 that were isolated between February 1982 and December 1983. Little or no difference was observed in the genomes of the same group, while the difference was evident between two groups. The Aichi/1/81 strain isolated in November 1981 had a genome distantly related to either of these two groups. Thus three different types of influenza C virus were isolated during the period of 12 mth from March 1981 to February 1982, suggesting that multiple influenza C viruses with distant genetic relationship were circulating at the same time in Japan.

Proteolytic activation of hemolysis and fusion by influenza C virus

SummaryInfluenza C virus has been found to cause pH-dependent hemolysis and fusion of chicken erythrocytes. For these activities, treatment of the virus with proteolytic enzymes, e.g., trypsin and elastase which were known to cause cleavage of gp88 was specifically required.

The functions of oligosaccharide chains associated with influenza C viral glycoproteins. II. The role of carbohydrates in the antigenic properties of influenza C viral glycoproteins.

SummaryThe antigenic properties of influenza C viral glycoprotein gp88 were compared with those of its nonglycosylated counterpart T76 synthesized in infected cells treated with tunicamycin. Radioimmunoprecipitation experiments with three different monoclonal antibodies against gp88 revealed that an antibody designated Q-5 precipitated gp88 but not T76, indicating the requirement for glycosylation for the binding of this antibody to gp88. It is unlikely, however, that the antigenic determinant recognized by Q-5 is carbohydrate moiety since the ability of the antibody to bind to gp88 varied depending on the virus strain, and trypsin-treatment of gp88 eliminated its reactivity with Q-5. Gel electrophoretic analysis under nonreducing conditions showed that T76 underwent the formation of disulfidelinked multimers in the absence of reducing agent while gp88 behaved as monomers, suggesting that glycosylation is required for gp88 molecules to attain an appropriate conformation. These observations, altogether, suggests that glycosylation is important in determining the immunological specificity of gp88 presumably by influencing the folding of this glycoprotein.

Intermolecular association of HANA glycoprotein of Sendai virus in relation to the expression of biological activities

Abstract When purified HANA glycoproteins of Sendai virus were centrifuged in a sucrose density gradient in the presence of Triton X-100, neuraminidase activity was distributed into two peaks, a rapidly sedimenting peak H and a slowly sedimenting peak L. The analysis of polypeptides under nonreducing condition showed that peak H contained HANA glycoproteins exclusively in the form of tetramer whereas peak L was in the form of dimer, which suggests that the different sedimentation rate is due primarily to the difference in the mode of intermolecular association of HANA glycoproteins by disulfide linkage. A glycoprotein designated C-HANA with a molecular weight of ∼55,000 was also involved in the solubilized HANA preparation and found as tetramer and dimer in peaks H and L, respectively, when analyzed under nonreducing condition. The C-HANA protein was produced only after solubilization of Sendai virions with Triton X-100. The C-HANA was found more in the preparation from egg-grown virions than from LLC-MK2 cell-grown virions, indicating that production of C-HANA is host cell-dependent. Trypsin treatment of the intact HANA oligomers resulted in the formation of oligomers of the size similar to the C-HANA oligomers. These results suggest that host cell-derived protease(s) present in the purified virus preparation is responsible for the cleavage. In contrast with the intact HANA oligomers, the C-HANA oligomers could neither reconstitute large multivalent structures nor exhibit hemagglutinating activity when centrifuged in sucrose density gradients in the absence of Triton X-100. Though the C-HANA oligomers could adsorb to chicken red blood cells, they were released rapidly from the cells even at a low temperature like monovalent forms of the intact HANA oligomers, suggesting that the formation of multivalent structure is essential for HANA glycoproteins to continue to adsorb to the cells and the C-HANA protein loses the hydrophobic part required for the reconstitution of HANA glycoprotein. In the light of these observations the previous result obtained by several authors who demonstrated an apparent dissociation of hemadsorbing activity from neuraminidase activity of paramyxoviruses can be reasonably explained now.