Yoshiyuki Nagai

Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus.

Abstract In search of a molecular basis underlying the variations in virulence observed with different strains of Newcastle disease virus, a comparative study has been carried out on the biosynthesis and function of the viral glycoproteins. Five virulent (Italien, Herts, Field Pheasant, Texas, Warwick) and five avirulent strains (La Sota, B 1 , F, Queensland, Ulster) have been analyzed. They were grown in five different host systems (embryonated eggs, cultures of BHK21-F, MDBK, chick embryo, and chick chorioallantoic membrane cells). Glycoprotein F (MW 56,000) which is responsible for hemolysis and cell fusion has been found with all strains to be derived by proteolytic cleavage from the precursor glycoprotein F o (MW 68,000). With strains Queensland and Ulster, in addition, a precursor glycoprotein HN o (MW 82,000) has been identified which is converted, again by proteolytic cleavage, into the hemagglutinin-neuraminidase glycoprotein HN (MW 74,000). Cleavage of F o is necessary for the expression of cell fusing and hemolytic activity, and the available evidence suggests that cleavage of HN o is paralleled by an enhancement of hemagglutinating and neuraminidase activity. However, activation of the glycoproteins is not required for virus assembly. Thus, virus particles containing the precursor F o may be formed which have a reduced infectivity. Infectivity is even lower, if both glycoproteins are present in the uncleaved form. After in vitro treatment with trypsin, such particles display full biological activity. Whether the glycoproteins are cleaved in vivo depends on the virus strain and on the host cell. With virulent strains, cleavage occurs in all host systems analyzed, and the virions formed contain HN and F. With avirulent strains, however, this is the case only in the embryonated egg and in cultures of chorioallantoic membrane cells. All other cells produce particles containing uncleaved glycoproteins. From these observations the following conclusions can be drawn: only a few host systems are permissive for avirulent strains, i.e., they produce highly infectious virus; other systems are nonpermissive for these strains, i.e., they produce defective virus; in contrast, all host systems studied are permissive for virulent strains. This concept is supported by the finding that multiple replication cycles and plaque formation occur only in permissive cells or in a nonpermissive culture after substitution of trypsin. Thus, plaque assays are now available for avirulent strains, either by the use of MDBK and chick embryo cells in the presence of trypsin or by the use of chorioallantoic membrane cells. These observations demonstrate striking differences in host range between virulent and avirulent strains which are determined by the susceptibility of the envelope glycoproteins to proteolytic cleavage. It is suggested that these differences account at least in part for the variations in the virulence of Newcastle disease virus.

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN0 and F0, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN0 and F0 together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN0 was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN0 and F0 with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN0 can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F0 shows a specific requirement for trypsin.

Studies on the assembly of the envelope of Newcastle disease virus

Abstract The association of the envelope proteins of Newcastle disease virus with membranes of infected BHK 21-F cells and their incorporation into mature envelopes has been investigated in a study employing cell fractionation. The principal fractions obtained by sucrose density gradient centrifugation of cytoplasmic extracts were rough endoplasmic reticulum and smooth membranes derived predominantly from smooth endoplasmic reticulum and Golgi apparatus. Furthermore, by adsorption to red blood cells it was possible to isolate virions and a hemadsorptive fraction of smooth membranes believed to be immediate precursors of mature envelopes. In addition to the cytoplasmic fractions, plasma membranes obtained as cell ghosts have been analyzed. Each fraction showed a distinct pattern of virus-specific proteins. Pulse-chase experiments indicated that glycoprotein HN and Fo were synthesized on the rough endoplasmic reticulum and transferred from there via smooth intracellular membranes to the plasma membrane and into virions. In the course of migration, Fo is converted to F. In contrast to the glycoproteins, protein M was found to be incorporated into the plasma membrane immediately after synthesis. Pulse-chase experiments also demonstrated that this protein appears in the hemagglutinating fraction of smooth membranes and in mature virions more rapidly than the glycoproteins. These results suggest that M is incorporated into membranes that contain already viral glycoproteins and that this process is one of the last steps in envelope assembly.

The Structure and Function of Paramyxovirus Glycoproteins

Most of the information on the structure, function and replication of paramyxoviruses has been obtained from studies on Newcastle disease virus (NDV), simian virus 5 (SV5), and Sendal virus (see reviews by Kingsbury, 1973; Choppin and Compans, 1975; Rott and Klenk, 1976). The virion consists of a helical ribonucleoprotein, the nucleocapsid, which is surrounded by an envelope (Fig.l). The nucleocapsid is composed of the single stranded RNA genome and a carbohydrate-free polypetide subunit, the NP protein. The envelope contains lipid which is derived from the plasma membrane of the host cell (Klenk and Choppin, 1969) and is present in the form of a bilayer (Landsberger et al., 1971). Associated with the outer side of the bilayer are spikes which are composed of glycoproteins (Klenk, et al., 1970; Chen, et al., 1971), the inner side is coated by the carbohydrate-free M protein. There are 2 types of spikes: one type consists of glycoprotein HN which has hemagglutinating and neuraminidase activity (Scheid et al., 1972; Scheid and Choppin, 1973; Seto et al., 1973;Tozawa, et al., 1973; Shimizu, et al., 1974), and the other one consists of glycoprotein F which induces cell fusion and hemolysis (Homma and Ohuchi, 1973; Scheid and Choppin, 1974; Seto, et al., 1974). It is generally agreed that these activities reflect the essential roles which both

Sendai Virus Biology and Engineering Leading up to the Development of a Novel Class of Expression Vector

Sendai virus (SeV), a prototypic member of the family Paramyxoviridae, was discovered in 1953, six decades ago. It is not just an old mouse pathogen but has been an irreplaceable model in in basic research to understand paramyxovirus replication and pathogenesis. The SeV reverse genetics established in 1996 has played a particularly prominent role in this context by settling outstanding issues and resolving enigmas. At the same time, the technology is evolving into a multipurpose cytoplasmic (nonintegrating) RNA vector. Its diverse medical applications are now in the pipeline and being tested in clinical settings as illustrated in the subsequent chapters. The production of diverse target-oriented devices has been possible by making full use of a variety of SeV theories and traits discovered during the six decades. Here, we summarize the long journey of SeV research leading up to the invention of this novel class of expression vector, SeV vector.