Richard W. Compans

Characterization of temperature sensitive influenza virus mutants defective in neuraminidase

Abstract Two temperature sensitive mutants, ts3 and ts11, of the WSN (HON1) strain of influenza virus, belonging to the same recombination group, have a 1000- to 10,000-fold lower infectivity titer and lack hemagglutinating and neuraminidase activity when grown at nonpermissive temperature (39.5°), compared with virus grown at permissive temperature (33°). The patterns of viral polypeptides synthesized in cells infected with these mutants are similar to those found with wild type virus. Neuraminidase activity of the mutant viruses is more temperature labile than the enzyme of the wild type. Despite the low yield, morphologically intact virus particles are found at 39.5°, but in contrast to virus grown at 33° large aggregates of virus accumulate near the cell surface. These aggregated virus particles contain neuraminic acid as demonstrated by a colloidal iron stain. Thus, it is likely that a neuraminic acid containing protein serves as receptor for the hemagglutinin of other virus particles, resulting in the extensive aggregation. Hemagglutinating activity of virus grown at 39.5° is restored by treatment of the aggregates with neuraminidase from Vibrio cholerae. These results suggest that the lack of hemagglutinating activity of mutant virus grown at 39.5° is a consequence of the formation of aggregates of virus particles carrying neuraminic acid on their surface, and that the ts defect is in the neuraminidase but not the hemagglutinin molecule. It is postulated that neuraminidase is essential for the replication of influenza viruses and is required to remove neuraminic acid from the viral envelope to avoid aggregation of the progeny virus.

Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action.

The neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) inhibits the mutlicycle replication of influenza viruses in tissue culture. Influenza virus grown in the presence of FANA contains neuraminic acid on its envelope which then serves as receptor for other virus particles causing extensive aggregation. Thus, FANA inhibits influenza virus replication by preventing the enzymatic removal of neuraminic acid from the virus envelope.

Influenza virus proteins: I. Analysis of polypeptides of the virion and identification of spike glycoproteins☆

Abstract The polypeptides of influenza virions have been analyzed by polyacrylamide gel electrophoresis. Seven polypeptides were detected in purified virions grown in three different cell types, and four of these polypeptides appear to be covalently linked with carbohydrate. Virions grown in different cell types were compared by coelectrophoresis, and host-dependent differences were detected in the electrophoretic mobility of some glycoproteins, but not of nonglycoproteins, suggesting that the carbohydrate moiety of such glycoproteins is specified by the host cell. The molecular weights of the seven proteins, estimated by coelectrophoresis with marker proteins, ranged from 83,500 to 26,500, with a total molecular weight of 380,500. Treatment of virions with the protease bromelain degraded the viral spikes resulting in particles which were bounded by a smooth-surfaced membrane, and which could be purified in a potassium tartrate gradient. Such particles were lacking three of the four glycoproteins; the four remaining viral proteins were unaltered by the enzyme. The particles devoid of spikes were noninfective, and they lacked hemagglutinin and neuraminidase activities, indicating that these properties are associated with the spike glycoproteins.

Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein

A method has been developed for the isolation of the glycoproteins of the parainfluenza virus SV5 using the nonionic detergent Triton X-100. Full recovery of hemagglutinating and neuraminidase activities was obtained. By rate zonal centrifugation in sucrose gradients containing 1% Triton X-100 and 0.5 M or 1 M potassium chloride, it was possible to separate the two glycoproteins. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 2, was 8.9 S and that of the smaller glycoprotein, virus protein 4, was 6.7 S. Each of the proteins aggregated when the detergent and KCl were removed, and the appearance of the aggregates differed with the two proteins. Both hemagglutinating and neuraminidase activities were found to be associated with protein 2; protein 4 exhibited neither activity. The results suggest that in this paramyxovirus both hemagglutinating and neuraminidase activities reside on a single glycoprotein. The biological function of the smaller SV5 glycoprotein remains to be determined.

Influenza virus structural and nonstructural proteins in infected cells and their plasma membranes

Abstract The virus-specific proteins in three types of cultured cells infected with the WSN strain of influenza virus have been analyzed. The seven structural proteins of the WSN virion were identified in infected cells, and in addition a nonstructural protein, designated NNP, was found in large amount in the nucleus of the infected cell. This nonstructural protein has an estimated molecular weight of ∼25,000 and is slightly smaller than protein 7, the smallest viral structural protein. Proteins NNP and 7 were isolated on an agarose column and shown to be distinct by peptide mapping. The seven viral structural proteins have been found in association with the plasma membrane of infected cells. Glycoproteins 5 and 6 are the last proteins to appear at the plasma membrane. The available evidence suggests that protein 2, which is found in much larger amount in infected cells than in the mature virion, is cleaved at the plasma membrane to yield proteins 5 and 6.

Reproduction of Paramyxoviruses

The paramyxovirus group is a large one which includes the parainfluenza viruses types 1–5, Newcastle disease, and mumps viruses. Measles, canine distemper, and rinderpest viruses form a distinct subgroup on the basis of antigenicity, hemagglutinating characteristics, and lack of evidence for a virion-associated neuraminidase or neuraminic acid-containing cellular receptors. However, it is now generally accepted that these viruses should also be included in the paramyxovirus group because of their similar structural properties. Other more recently isolated viruses which have been classified as paramyxoviruses on the basis of morphological and biological properties are Yucaipa (Dinter et al., 1964) and Nariva (Walder, 1971) viruses. Table 1 lists paramyxoviruses and their primary hosts.

An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5☆☆☆

Abstract The multiplication of the parainfluenza virus SV5 was studied in primary rhesus monkey kidney (MK) cells and in a line of baby hamster kidney (BHK21-F) cells. Virus adsorbs to the cell surface and appears to penetrate by phagocytosis. Virus morphogenesis is similar in the two cells; the helical nucleocapsid forms in the cytoplasmic matrix and aligns under regions of cell membrane which acquire viral surface projections. Assembly and release of virus particles at the cell surface occurs by a budding process involving the incorporation into the viral envelope of a unit membrane which is continuous with and morphologically identical to that of the host cell. Both spherical and filamentous virus particles are formed; filaments frequently contain regularly coiled nucleocapsid throughout their length. In MK cells, which yield high titers of infective virus for many days but show little cytopathic effect, a balance appears to exist between synthesis of nucleocapsid and its continuous release within mature virus particles. In contrast, in BHK21-F cells, which produce little virus and disintegrate after extensive cell fusion, large aggregates of nucleocapsid accumulate in the cytoplasm, suggesting a block in maturation at the cell membrane. The present electron microscopic observations support previous studies which suggested that the virulence and yield of SV5 may depend, at least in part, on the response of the cell membrane to the virus.

Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide HA.

Abstract The cleavage of the HA polypeptide, the largest glycoprotein of influenza virus, to polypeptides HA1 and HA2 has been studied using the WSN strain of influenza A 0 and the RI/5 − strain of influenza A 2 grown in different host cells. Cleavage of the HA polypeptide is not required for the assembly of infectious, hemagglutinating virions. Cleavage is both strain dependent and host cell dependent, and correlates with the extent of cell damage, suggesting that the enzymes involved are host cell specified. Virions grown in MDBK cells without calf serum in the medium contain almost entirely uncleaved HA polypeptides. Virions harvested early in the growth cycle contain more uncleaved HA polypeptide than virions harvested late from the same cells when cytopathic effects are extensive. The proteolytic nature of the cleavage has been demonstrated in vitro with trypsin. Comparison of the specific hemagglutinating activity and infectivity of virions which contain different amounts of the uncleaved HA polypeptide and the cleavage products HA1 and HA2, and of the capacity of such virions to react with the soluble glycoprotein receptor substance fetuin, have shown that uncleaved HA polypeptides are as active in hemagglutination and adsorption to cellular and soluble receptors as are the disulfide-bonded complexes composed of HA1 plus HA2. The proteolytic cleavage of the HA polypeptide to HA1 and HA2 is thus not required for virus assembly or for full expression of the biological properties of the virion. Rather, it appears to be a nonessential result of events occurring in infected cells which are undergoing cytopathic effects.

Influenza virus proteins: II. Association with components of the cytoplasm

Abstract Cytoplasmic extracts of influenza virus-infected BHK21-F and MDBK cells were separated by equilibrium sedimentation into fractions containing smooth membranes, rough membranes, and free ribosomes and polysomes. Analysis by polyacrylamide gel electrophoresis revealed that viral polypeptides were associated with all cytoplasmic fractions. Smooth membranes contained large amounts of viral glycoproteins, as well as the nonglycosylated polypeptide (M) thought to be associated with the viral membrane. Rough membranes also contained the uncleaved hemagglutinin glycoprotein (HA), and results of pulse-chase experiments suggest that this polypeptide is synthesized in association with the rough membranes and accumulates in smooth membranes. The nucleoprotein subunit NP was located mainly in the soluble fraction as well as a cell fraction of intermediate density. The nonstructural viral polypeptide NS (∼25,000 MW) appeared to be associated specifically with ribo-some-containing fractions.

Reproduction of Myxoviruses

The myxovirus group consists of the influenza viruses of man and animals. The designation was originally suggested (Andrewes et al., 1955) for a group of viruses which exhibited affinity for certain mucoid substances, and included mumps and Newcastle disease viruses (NDV) in addition to influenza viruses. It was recognized subsequently that the former two viruses should be separated into a distinct group because of numerous differences in structure and replication between them and the influenza viruses (Waterson, 1962), and the term paramyxovirus has been adopted for the group which includes these agents. Thus, the myxovirus group (sometimes referred to as orthomyxoviruses; Melnick, 1973) as presently constituted contains only the influenza viruses. As described in sect. 2, among the polypeptides of the influenza virion are two distinct surface polypeptides with hemagglutinin and neuraminidase activities and an internal ribonucleoprotein (RNP). The antigenicity of these polypeptides has provided a basis for the classification of influenza viruses into types, subtypes, and strains. Table 1 indicates the system of classification and lists some of the strains in common use.