Masao Iinuma

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.

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

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.

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.

Cross-linking of Newcastle disease virus (NDV) proteins

SummaryThe proxomity and spatial relationships of the structural proteins of Newcastle disease virus (NDV) were studied by chemical cross-liking with a series of imidoesters. When the virions were reacted by the cross-linker with a distance 6.1 Å or longer between the functional groups and analyzed by polyacrylamide gel electrophoresis, remarkable changes were observed in the migration patterns of the viral proteins. The most striking one was the extensive decrease in the intensity of the M protein band, and although not so strikingly, glycoprotein and nucleocapsid protein bands were reduced significantly. Instead, several protein complexes appeared at and near the top of the gels. The protein complexes formed by a reversible cross-linker, dimethyl-3,3′-dithiobispropionimidate (DTBP), were analyzed by two dimensional electrophoresis; the complexes on the first-dimension cylindrical gels were cleaved by reduction with 2-mercaptoethanol and electrophoresed laterally on the second-dimension slab gels. The results indicated that homodimers of glycoprotein, nucleocapsid protein and M protein were generated under the condition of the most gentle cross-linking employed. At the same time, however, trimer and higher homopolymers of M protein were already detectable. Under the more extensive conditions, the bulk of M protein was cross-linked to form a large protein complex with very high molecular weight. Further, small but significant amounts of glycoprotein and nucleocapsid protein were always detected in this complex.These results suggest that M protein may be present in the virion in close enough proximity to interact with each other and may further have some interactions with glycoprotein and nucleocapsid protein. On the basis of these findings possible roles of M protein in virus assembly were discussed.

Studies on the assembly of Newcastle disease virus: incorporation of structural proteins into virus particles

Summary The mechanism of assembly of Newcastle disease virus in chick embryo cells was investigated in two series of experiments. When protein synthesis was inhibited by addition of puromycin, cycloheximide or fluorophenylalanine at any time during the course of infection, subsequent virus production was soon inhibited. These drugs were inhibitory even when they were added to the cultures as late as 10 hr after infection, when large amounts of virus precursor proteins were present within the cell. In the second series of experiments, the kinetics and efficiency of incorporation of radioactive amino acid into virus particles were examined by the pulse-labelling technique. A 30 min. labelling period at the 3rd or 6th hr of infection resulted in the release of highly radioactive virus during the period of 1½ hr immediately after the pulse. However, when pulse-labelling was performed at the 9th hr, the maximally labelled virus was found in the yield obtained 3 hr after the pulse, and the specific radioactivity of virus was less than 1% of that of the virus harvested from cultures labelled earlier. On the basis of these findings, possible mechanics of virus assembly are discussed.

Proteins of Newcastle disease virus. A comparison by partial protease digestion among the strains of different pathogenicity.

Abstract To investigate the relationship between the pathogenicity of Newcastle disease virus and the structure of viral proteins, two typical strains were sampled from each of three groups of different pathogenicities and these six strains were compared for protein structure by sizing peptides generated by partial digestion with Staphylococcus aureus V8 protease and chymotrypsin. These digests yielded closely similar peptide patterns for the internal polypeptides L and NP, whereas those of the glycoproteins HN and F showed apparent variations which appeared to be specific for the individual groups. Although not as significant as in the glycoproteins, group-dependent variations were also detectable with the M protein. These results suggest that the external proteins might undergo considerable changes whereas the internal proteins would be highly stable and that there is a definite correlation between such changes in the external proteins and the pathogenicity of the virus.

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p‐fluorophenylalanine (FPA) was added to influenza virus RI/5+‐infected cells 4 hr after infection, virus‐specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA‐treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.