Toshisada Matsumoto

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.

Studies on the role of M protein in virus assembly using a is mutant of HVJ (Sendai virus)

Abstract A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F 0 , NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F 0 , and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.

The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins.

The pathogenic strain Italien and the apathogenic strain Ulster of Newcastle disease virus have been compared with respect to organ tropism and spread of infection in 11-day-old chick embryos. After infection of the endodermal layer of the chorioallantoic membrane by intra-allantoic inoculation with strain Italien, high virus titres are found in all extra-embryonic membranes and fluids and in the embryo itself. Infection results in early death of the embryo. In contrast, after infection with strain Ulster by the same route of inoculation, high virus titres are found only in the allantoic sac and embryos are not killed. Inoculations with strain Italien on to the ectodermal layer through an artificial air sac results in rapid spread of infection in the chorioallantoic membrane and the embryo dies before the virus invades other tissues including the embryo. Under the same conditions of infection, strain Ulster neither spreads within chorioallantoic membrane nor does it kill the embryo. Virus spread in each germinal layer of the chorioallantoic membrane was analysed by immune fluorescence. These studies showed that endoderm as well as mesoderm and ectoderm allowed the spread of strain Italien, whereas only the endoderm is permissive for strain Ulster. These differences in host range are based upon differential activation of the virus glycoproteins by proteolytic cleavage. The glycoproteins of strain Italien are cleaved in each germinal layer, whereas those of strain Ulster are cleaved only in endoderm. These studies demonstrate that, in the system analysed here, spread of infection and organ tropism are important factors for pathogenicity and both of these factors are determined by the susceptibility of the virus glycoproteins to proteolytic cleavage.

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.

The polypeptides of mumps virus and their synthesis in infected chick embryo cells

Abstract The polypeptides of mumps virus grown in chick embryo (CE) cells were analyzed by polyacrylamide gel electrophoresis and partial proteolytic mapping. The virus contains five unique polypeptides, gp80(HN), p73(NP), gp61(F1), p45, and p39, and an additional few minor species, p71, p47, and p42. Except for p45 and p42 they were remarkably phosphorylated. The polypeptide p71 was related to p73 and these two proteins comigrated on the gels in the presence of urea. There was also a close relationship between p47 and p45 and the former was a phosphorylated form of the latter. The polypeptide p42 was suggested to be actin derived from the host cell. Under nonreducing conditions of electrophoresis, the glycoprotein F(gp73) was identified, which consisted of two disulfide bonded glycopeptides, F1 and F2(gp10). The development of viral proteins in infected CE cells was analyzed by immunoprecipitation followed by polyacrylamide gel electrophoresis. It was demonstrated that p45 was synthesized very early in infection and rapidly reached the maximal rate of synthesis. In contrast, the appearance of the other polypeptides was delayed and their synthesis rates were found to increase with time until a very late stage. Thus the patterns of early and late protein synthesis were largely different from each other, suggesting a distinct temporal regulation in the mumps virus protein synthesis. Pulse-chase experiments demonstrated that the glycoprotein F would be formed by proteolytic cleavage of a precursor glycoprotein F0 with an apparent molecular weight of 73,000. No other proteolytic processing has been found.

A host-induced modification of hemagglutinating virus of Japan (HVJ, Sendai virus) in its hemolytic and cytopathic activity☆☆☆

Abstract Host-induced changes of HVJ in hemolytic activity and in growth characteristics in mouse lung cells were investigated. HVJ grown in the allantoic cavity of the chick embryo is hemolytic, but the virus loses this property after a single passage in the mouse lung or after a single growth cycle in cultivated mouse lung cells. This nonhemolytic virus becomes hemolytic after a single chick embryo passage. Similar changes in virus growth characteristics were also observed. When HVJ was adapted to growth in monolayers of mouse lung cells, virus was produced within 24 hours of infection, but cytopathic effects were delayed, often for several days. After one passage in the chick embryo, however, this virus reproduced in the tissue culture with concomitant destruction of the cells. After a single passage in tissue culture the cytopathic virus returned to the noncytopathic condition.

Subunits of NDV: Hemagglutinin and neuraminidase subunits of Newcastle disease virus☆

Abstract When purified Newcastle disease virus (NDV) was treated with deoxycholate (DOC), two major surface antigens of the virus, hemagglutinin and neuraminidase, were released in biologically active form and the respective antigens could be separated by sucrose gradient centrifugation into two distinct bands. The hemagglutinin subunits obtained by the DOC method were reactive with hemagglutination-inhibiting antibody but did not agglutinate red blood cells even after they were freed from DOC by dialysis. Thus, they appeared to be monovalent. However, they acquired the capacity to agglutinate red blood cells, when they were mixed with the top fraction of the sucrose gradient through which the DOC-disrupted virus had been centrifuged, or by the addition of a phospholipid, phosphatidylethanolamine. Electron microscopic examination of the hemagglutinin subunits revealed filamentous structures associated with several projections on both sides. These results suggested that polymerization of the monomeric subunits occurred, not directly end to end, but mediated through the filament which might be phospholipid in nature.

The Pathogenicity of Newcastle Disease Virus Isolated from Migrating and Domestic Ducks and the Susceptibility of the Viral Glycoproteins to Proteolytic Cleavage

Besides the chicken, other poultry are known to be susceptible to Newcastle disease virus (NDV) (9), and the virus appears to be distributed widely in various wild birds as indicated by isolation of new NDV strains from such birds (10, 11). We considered it to be of ecological interest to learn the properties of such virus strains since there is a possibility that some species of wild birds act as a reservoir from which the infection in fowls arises. Recently evidence that pathogenic and apathogenic strains of NDV differ from each other with respect to the susceptibility of the viral glycoproteins to proteolytic cleavage was obtained (4, 6). The glycoproteins of pathogenic strains are readily cleaved to become biologically active molecules in all types of host cells tested, whereas those of apathogenic strains are cleaved only in the endoderm of the chorioallantoic membrane of the chick embryo but not in most other cells. On the basis of these studies we have characterized five new NDV strains, D-13, D-26, D-28, D-43, and D-49, which were recently isolated from various migrating and domestic ducks in Japan by Yamane et al (11). The stock viruses were grown in the allantoic cavity of 11-day-old chick embryos. The capacity of the viruses to kill chick embryos was determined by measuring the mean death time (MDT) of the embryo after inoculation of 102.5 EID50 of the viruses into the allantoic cavity of 11-day-old chick embryos (9). Labeling of virus with [3H]-glucosamine (New England Nuclear Corp., 10-30 Ci/mmol) in MDBK cells and isolation of the labeled virions were carried out as described previously (4, 5). To analyze proteins and glycoproteins, virions were dissociated with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol and subjected to electrophoresis on polyacrylamide slab gels in Tris-glycine buffer with SDS (2). [3H]-glucosamine on the gels was detected by scintillation autography (1). Hemagglutinin, neuraminidase and hemolytic activities were determined as described previously (4). Infectivity titers were determined by infective doses for MDBK cells as indicated by the production of detectable amounts of hemagglutinin (8). First we examined the virulence of the newly isolated strains for chick embryos by determining the MDT. All of the five strains were found to have an MDT of

Relation of interferon production to the limited replication of Newcastle disease virus in L cells.

Growth of Newcastle disease virus (NDV) in L cells, where the virus undergoes limited replication, has been compared to that in fully permissive BHK-21 host cells. The synthesis of viral proteins and the production of infectious progeny were found to occur normally at early times of infection in L cells. However, the subsequent amplification of viral protein synthesis did not take place and instead of infectious virions, non-infectious haemagglutinin was the predominant product. This shift of replication pattern from complete to incomplete virus production seemed to be temporally related to the appearance and accumulation of interferon (IFN) in the system. The addition of specific antiserum against mouse IFN to the infected L cell cultures was able to circumvent such restriction of virus growth. In the presence of the antiserum, synthesis of viral proteins was found to progress normally, with the production of a high amount of infectious progeny comparable to that of the permissive system. These results suggest that the limited replication of NDV in L cells may be due to interference by the endogenously produced IFN during the course of infection.

Studies on the assembly of Newcastle disease virus: an arginine-dependent step in virus replication.

Abstract Growth of Newcastle disease virus in HeLa cells deprived of various amino acids was investigated. Of all amino acids in Eagles MEM, only arginine was found to be essential for the synthesis of infectious progeny virus. Omission of any one or all of the other amino acids from the culture medium permitted limited virus production, but none whatever could be detected in the absence of medium-arginine. Although the arginine-deprived cells had synthesized a large quantity of nucleocapsid antigen, hemagglutinin and neuraminidase at the later stage of infection, neither hemadsorption on the cell surface nor release of infectious virus could be detected. When arginine was restored to the culture, hemadsorption reaction and release of virus became soon detectable and the virus titer increased rapidly. Simultaneous addition of cycloheximide with arginine did not result in such a recovery of virus production. Immunofluorescent staining of NDV-infected cells with antiviral antibody showed that the cells incubated in normal medium exhibited clear fluorescence at the cell surface, but the surface of arginine-deprived cells did not show any detectable fluorescence, whereas either cells showed distinct cytoplasmic fluorescence when stained after fixation. These results suggest that arginine plays a specific role in the synthesis of a protein which might be an essential for virus maturation.