- Volume 21, Issue 2, 1973

Newcastle disease virus (NDV) or Sendai virus added to washed suspensions of rabbit or human platelets induced a brisk platelet aggregation (P.A.) response concomitant with release of biologically active components from storage granules (platelet release reaction, P.R.). NDV which had been rendered non-infectious by u.v. irradiation for 5 min retained its capacity to induce P.A. and P.R. and to agglutinate or lyse erythrocytes. Treatments of paramyxoviruses (heating at 56 °C; disruption by Tween 80/ether) which resulted in loss of haemolytic activity also eliminated P.A. and P.R. reactions but had little or no effect on virus haemagglutinating properties. A non-haemolytic virus, influenza A2, had no detectable effect on platelets when used at a concentration having a haemagglutination titre equivalent to that of platelet-aggregating doses of NDV or Sendai virus.

It was concluded that the properties of paramyxoviruses responsible for causing lysis of erythrocytes were required for induction of P.A. and P.R. reactions. In this respect, the virus-induced platelet responses may be similar to other cellular reactions to paramyxoviruses such as fusion of cell membranes and development of cytotoxic or cytopathic changes.

Wild-type poliovirus (ts +) rapidly represses synthesis of host cell protein (psr + character), at permissive (37 °C) or restrictive (39.5 °C) temperatures. A search for ts mutants that were psr + at 37 °C and psr at 39.5 °C was successful when gene expression was minimized by adding guanidine, depleting cystine and avoiding very high input multiplicities. Repressor defects were found in six ts mutants, all of which carry ts defects solely in structural protein. All of the five ts mutants tested that were defective in non-structural genes failed to show a repressor defect. The repressor defect was confirmed to lie in structural genes by showing that three out of four independent ts + revertants from a structural protein (psr. ts) mutant were also psr +. Thus repression is dependent on the configuration of a product of the structural protein gene. These findings are discussed in terms of the equestron, a hypothetical poliovirus regulator.

We have isolated temperature-sensitive mutants of SV 40 in the lytic system by mutating the virus with nitrous acid or hydroxylamine, and characterized 11 of them. Nine mutants were grouped into two complementation classes in the productive infection of CV-1 cells. The first group contains a single mutant, which was defective in synthesizing virus DNA at the restrictive temperature, but induced the synthesis of cellular DNA. The mutants of the second class produced normal amounts of virus DNA and V-antigen at the restrictive temperature. A naturally-occurring large plaque strain belonged to this group. Both groups retained the transforming ability at the restrictive temperature. Two mutants showed no complementation with either group. One of them had the characters of the first group. The other mutant did not show temperature-sensitivity when the growth was initiated by infection with its DNA. This mutant did not transform 3T3 cells even at the permissive temperature. Rabbit kidney cells, however, were transformed by this mutant effectively at the permissive temperature but less at the restrictive.

The growth of alastrim virus in chick embryo cells was studied under one-step conditions at various temperatures. Limited growth was found at 37 °C but there was none at 38 °C or above. Temperature-shift experiments suggested that at 38 °C only late events were temperature-sensitive. Virus DNA synthesis, induction of early enzymes and the production of early antigens were all substantially normal at 38 °C. In a study of late events at 38 °C, particle formation was found to be almost completely inhibited. Although a few immature particles were seen by electron microscopy of thin sections, cytoplasmic DNA labelled with [3H]-thymidine did not become resistant to DNase and particles containing DNA were not seen after centrifuging on sucrose density gradients. There was no late rise in DNA-dependent RNA polymerase activity. Late antigen production at 38 °C appeared normal in agar gel diffusion studies both in the time of appearance and in the number of lines present, but the LS-antigen complex was slightly reduced in amount. Production of haemagglutinin was completely suppressed at 38 °C. It is concluded that the major effect of temperature on the growth of alastrim virus is to inhibit a very early stage in particle formation.

KB cells infected with rhinovirus type 14 (RV14) in the presence of actinomycin D synthesized two major species of virus RNA. One species had properties similar to infectious RNA extracted from virus particles. The other major species of virus RNA exhibited properties that were descriptive of replicative intermediate (RI) structures. Pulse-labelling experiments with [3H]-uridine suggested that the RI structures were involved in synthesis of virus RNA. Two RNA species were isolated in minor amounts from infected cells. One species had properties descriptive of a replicative form of RNA. The second minor species was single-stranded, approximately one-half the size of the virus genome and could also be isolated from purified virus.

Cytopathological changes induced by carrot mottle virus in systemically infected cells of Nicotiana clevelandii plants grown at 17 °C and illuminated at 4000 lux for 8 h/day, were studied in relation to the infectivity of buffer and phenoltreated extracts. Changes were most obvious in palisade cells. About six days after inoculation, tubules appeared in the cytoplasm, associated with the plasmodesmata. Later the tubules, some of which became sheathed by cell wall material forming plasmodesmatal outgrowths, extended towards the vacuole, others towards the nucleus, causing invaginations in it. Endoplasmic reticulum and Golgi bodies increased, especially in areas adjacent to the nuclei, and complexes of cytoplasmic tubules with endoplasmic reticulum were observed. Membrane-bound particles, some with densely-staining central spots, appeared in the vacuole close to the tonoplast and reached their maximum number after 8 to 9 days. Sometimes they were clustered where a plasmodesmatal tubule or outgrowth came close to the tonoplast.

Tests with the phenol-treated and buffer extracts showed that the cells contained both labile and stable forms of infectivity. The former predominated early in systemic infection (about 5 to 7 days after inoculation) and then declined, while the amount of stable infectivity increased, reaching a maximum after 8 to 9 days. It is suggested that the labile form of infectivity consists of RNase-sensitive material, probably nucleocapsids, which become stable (RNase-resistant) when they receive a protective envelope on entry into the vacuole.

It has been shown by competition hybridization experiments that common sequences of about 600 nucleotides are present in TRV long and short particle RNA species. Heteroduplex molecules have been formed by the hybridization of short particle RNA strands with long complementary strands derived from tissue infected with a ‘defective isolate’ of the virus. The heteroduplex molecules were shown to have a lower melting temperature than either the long-long or short-short homoduplexes, indicating some mismatching of base pairs. The results are discussed in the light of our present knowledge about the replication of viruses.

Theoretical equations have been derived to describe RNA-RNA competition hybridization and duplex formation.

Reduced yields of poliovirus and foot-and-mouth disease virus are obtained in suspended cell cultures in the presence of rifampicin. The effect of the drug appears to be related to virus replication and not to a toxic effect on the host cells. The inhibition is reversed upon removal of the drug. The replication of both picornaviruses in suspended cultures is affected by actinomycin D, but not by mitomycin C.

Strains of Newcastle disease virus (NDV), which were good or poor inducers of interferon in chick cells, occurred in all virulence groups of this virus. Mesogenic and velogenic strains which killed the embryo rapidly were better interferon inducers in embryonated egg than were the lentogenic strains. Identical and low sensitivities to interferon were shown by an interferon-inducing vaccine strain and a non-inducing and highly virulent strain. Measurements made when various strains were used to infect either normal or interferon-pre-treated cells showed that the absence of interferon production was not due to the inhibitory effect of virus infection on cellular protein synthesis. It is suggested that infective NDV synthesizes two kinds of inhibitory protein during infection. One of these inhibits the synthesis of the interferon protein and in consequence the majority of strains do not induce interferon in chick cells. The second protein is induced mainly by mesogenic and velogenic strains and may be responsible for inhibiting cellular protein synthesis.

Barley yellow striate mosaic virus (BYSMV) [cryptogram: */*:*/*:U/*:S,I/Au] was detected in the cytoplasm but not in the nucleus or other organelles of infected barley cells. Stiff bacilliform particles of about 45 × 330 nm, believed to be the mature virus particles, occurred as membrane-bound aggregates while slightly flexuous particles of similar length and 35 to 45 nm wide, possibly representing ‘immature’ virus particles, were seen dispersed within sac-like structures. Such particles had sometimes an electron-transparent end and were associated with spherical vesicles 25 to 60 nm in diam.

Electron-dense intracytoplasmic masses of granular or finely fibrous material, here called viroplasm, were consistently found in connexion with BYSMV particles which were apparently formed by budding from them through membranes limiting the surrounding enclaves. These appeared to have derived from the endoplasmic reticulum and, occasionally, from the separation of the nuclear envelope lamellae. The number and size of BYSMV viroplasms, together with their close connexion with both the endoplasmic reticulum and the outer lamella of the nuclear envelope, seem to be features unique among the described plant rhabdoviruses.

The cytopathogenicity and production of Newcastle disease virus (NDV) strain Herts cultivated in chick embryo (CE), baby hamster kidney (BHK-21), HEp-2, MDBK and L929 cells was investigated. Infection at high multiplicities (1000 p.f.u./cell) induced cell fusion in cultures of all cell types within 3 h after infection. Infection at low multiplicities (10 to 20 p.f.u./cell) produced extensive cell fusion in CE, BHK-21, HEp-2 and L929 cultures within 24 h, but MDBK cells failed to fuse. The latter cells failed to show high levels of virus haemagglutinin at the cell surface or accumulation of virus products, but infective virus was released to significantly higher titres than from the cell types which fused. However, infected MDBK showed similar levels of virus-induced cell damage to the plasma membrane, as measured by the release of lactate dehydrogenase, to HEp-2 and L929 cells which were susceptible to fusion. The morphology of the c.p.e. produced by strain Herts in the different cell types is described. The relationship of virus accumulation and virus release to the process of cell fusion is discussed.

The replication of rabbitpox virus has been studied in mouse sarcoma 180 cells in media containing either arginine or citrulline. The yield of infective virus depended on the concentration of each amino acid, and maximum yield was obtained with 0.1 mm-arginine or 0.5 mm-citrulline. In the presence of this concentration of citrulline, cell multiplication and protein synthesis in uninfected cells was suppressed markedly compared with cell cultures in medium containing 0.5 mm-arginine. Growth of a human citrullinaemia cell line was inhibited also when citrulline substituted for arginine yet the replication of vaccinia virus continued.

The incorporation of radioactivity available as [14C]-carbamoyl-citrulline into mouse sarcoma 180 cells maintained in citrulline-containing medium increased significantly following infection by rabbitpox virus. A similar, increased incorporation was observed with vaccinia virus-infected HeLa cells. Increased incorporation into rabbitpox virus-infected mouse sarcoma 180 cells occurred at 3 and 5 h post-infection and coincided with increased incorporation of [14C]-guanido-arginine into cell cultures infected in the presence of arginine. This enhanced utilization of citrulline was inhibited by actinomycin D, and rabbitpox virus replication in the presence of citrulline was inhibited by canavanine. It is concluded that virus-directed mechanisms determine arginine biosynthesis in mouse sarcoma 180 cells infected with rabbitpox virus in the presence of citrulline.

Argininosuccinate synthetase-lyase activity in HeLa or mouse L cells increased markedly following infection by vaccinia virus. A repression mechanism determines specific activities in uninfected cells and greater activity was obtained in the presence of 0.2 mm-citrulline than of 0.6 mm-arginine. However, maximum activity in infected cells was obtained with 0.6 mm-arginine. The development of increased enzyme activity in virus-infected cells was inhibited by puromycin or actinomycin D. Treatment of infected cells with FUdR resulted in higher levels of activity than in similar cells in the absence of the inhibitor. The Michaelis constants of enzymes in HeLa and mouse L cells were 0.11 × 10−3 m and 0.07 × 10−3 m, respectively; the same value of 1.0 × 10−3 m was determined for both species of infected cells. Amino acid analysis of purified vaccinia virus prepared from HeLa cell cultures infected in the presence of [14C]-carbamoyl-citrulline showed that radioactivity was associated exclusively with arginine. It is concluded that the anabolism of arginine from citrulline in vaccinia virus-infected cells is determined by virus-coded enzymes.

Elongated protrusions (diam. 150 to 250 nm) extending from the cell membranes, have been observed by scanning electron microscopy and in thin sections of calf kidney cells 10 h after infection with high multiplicities of parainfluenza virus 3 (PIV-3).

Unfixed, flattened PIV-3 particles, were pleomorphic with diam. ranging from 100 to 500 nm. Following early fixation of fresh virus harvests, the particles appeared spherical in the scanning and transmission electron microscopes, and had a mean diam. of 135 nm. These virus particles had not been flattened on the support film as demonstrated by goniometer analysis.

Spontaneous segmentation of unfixed PIV-3 envelopes was observed. Phospholipase C digestion of the virus resulted in a similar segmentation, producing round envelope aggregates with extending peplomers.

This paper describes the physico-chemical properties of Nodamura virus. The virus infects mice and high yields (107 LD50/mg) were obtained from the muscle tissue. To allow for greater ease and accuracy in analysing the RNA, the virus was labelled by inoculating mice with [32P]-phosphate during the incubation period. Purified virus was obtained from the muscle extracts by ammonium sulphate precipitation, differential ultracentrifuging and sedimentation of the SDS-disrupted virus pellet in a sucrose gradient. The virus particles were 29 nm in diam., sedimented at approximately 135S in sucrose gradients and contained two species of RNA which sedimented at 22S and 15S. These values correspond to mol. wt. of 1.0 and 0.5 × 106. Similar mol. wt. were obtained by polyacrylamide gel electrophoresis. The base ratios of the two RNA species were: 22S: A = 22.4, C = 27.0, G = 27.6, U = 23.0; 15S: A = 24.8, C = 28.2, G = 22.8, U = 24.2. Each RNA had low infectivity in mice but this was enhanced about 100-fold by mixing the two species. The RNAs may be present in two distinct particles since extraction of the 135S peak with phenol gave the 22S RNA alone, whereas phenol-SDS extraction liberated both the 22S and 15S species. Centrifuging in caesium chloride also fractionated the virus into components which contained either the 22S or 15S RNA. Preliminary evidence suggests that there is one major polypeptide, mol. wt. 35 × 103, and two minor polypeptides in the virus.

Antigenic poliovirus receptor sites on mammalian cells were compared using an antireceptor serum. Antiserum to HeLa cells was found to block the attachment of enteroviruses to live HeLa cells. This blockage was made more specific by adsorbing the anticellular serum with homologous cells whose surface receptors for polioviruses had been selectively heat inactivated. The adsorbed homotypic serum inhibited specifically the attachment of polioviruses to live HeLa cells, while the receptors for coxsackievirus B3 remained functional. Treatment of FL, ML, U, AV-3 and CV-1 cells with this antireceptor serum also resulted in the specific blockage of the receptors for polioviruses but not the receptors for coxsackievirus B3. These results indicate that the surface receptors for polioviruses on various cell lines differ antigenically from the receptors for coxsackievirus B3, and that poliovirus receptors are antigenically homologous on different cell lines.

Mouse peritoneal macrophages from C57 Bl and NIH mice were examined after infection with polyoma virus. Cell DNA synthesis was stimulated in both cell types to the same extent between two and three days after addition of virus. Morphological changes appearing soon after infection were reversed by the second or third day. The cells did not acquire other properties associated with the transformed state. Treatment with mouse interferon up to 48 h after infection inhibited the virus-induced host DNA synthesis while morphological changes were not affected.

Treatment of foot-and-mouth disease virus with 4% glutaraldehyde increases the diam. of the particles by 25% and makes them permeable to phosphotungstic acid so that they appear empty. The treated particles also resemble naturally-occurring empty particles in their low sedimentation coefficient (about 75S) but, in contrast to empty particles, they have a normal content of RNA and a higher than normal buoyant density in caesium chloride. The RNA can be removed from fixed particles by ribonuclease. Two models are suggested which account for these alterations in the structure of the virus particles. These results show that fixation with glutaraldehyde, far from maintaining the structural integrity of the virus particles, leads to considerable alterations in the arrangement of the RNA and protein subunits.

The plasma membranes of BHK 21 cells infected with vesicular stomatitis virus react in complement-fixation tests with antiserum to uninfected BHK 21 cells and with antiserum to the glycosylated ‘spike’ protein found on the surface of the virus (protein G).

Depending upon the time after infection, the minor protein NS or the nucleoprotein N predominates in the cytoplasmic sap. During the synthesis of virus these two proteins are associated in the cell cytoplasm with the virus RNA in a complex having a similar sedimentation coefficient to that of the virus nucleocapsid (140 S).

The linkage of the nucleocapsid with the altered cellular membrane appears to be a function of the ‘matrix’ or ‘virus membrane protein’, protein M. In the BHK 21 system it has not been possible to demonstrate the intracellular presence of protein M although incubation of infected cells with [14C]-labelled amino acids produced labelled protein M in the extracellular virus. The possibility that the rate of formation of this protein controls virus production is discussed.