- Volume 31, Issue 3, 1976

Escherichia coli b/r (su 0) was infected, at 30 °C, with T4Dam +, T4DamB24amN82 (1 -, 44 -, DNA-negative phenotype), and T4DamN134amBL292 (33 -, 55 -, maturation-defective phenotype). A genetic (‘transformation’) assay was used to monitor transcription of genes 30 (polynucleotide ligase), 42 (deoxycytidylate hydroxymethylase), 43 (DNA polymerase), rIIA, rIIB, and e (endolysin). The principal results are: (1) All of the genes studied were transcribed exclusively from the so-called l-strand of phage DNA. (2) DNA synthesis and the maturation-defective proteins were required to turn-off transcription of genes 42, rIIA, tIIB, and 43. Experiments performed with chloramphenicol suggested that all phage-specific proteins required to turn-off transcription of these genes were not present until 6 to 8 min post infection (p.i.). (3) During a normal developmental programme, gene 30 was transcribed throughout the eclipse. DNA-negative and maturation-defective conditions had no obvious effect on transcription of this gene. (4) During a normal lytic event, two discrete waves of gene e transcription were observed. The late wave was dependent upon DNA-synthesis and presence of functional maturation-defective proteins. The early wave was unaffected by DNA-negative or maturation-defective conditions. Experiments with chloramphenicol indicated that, if any virus-specific proteins are involved with regulation of early e transcription, such proteins are present by 3 min p.i.

The data are interpreted to mean that early gene transcription is regulated by a minimum of two mechanisms. One of these mechanisms is fully operational by the 3rd min and, among the genes studied, controlled early e transcription. A second mechanism becomes operational between 6 and 8 min p.i. and controls transcription of genes 42, 43, rIIA, and rIIB.

Herpes simplex virus can confer to thymidine kinaseless cells the ability to incorporate exogenously supplied thymidine into acid precipitable material. However no incorporation of exogenously supplied deoxycytidine into acid precipitable material can be detected after infection of deoxycytidine kinaseless cells by herpes simplex virus.

This failure to incorporate exogenous deoxycytidine is not due to the failure of the deoxycytidine phosphorylating activity of the virus induced deoxypyrimidine kinase but to a block in the metabolism of deoxycytidine monophosphate in herpes simplex virus infected cells. This block becomes evident with the appearance of the virus induced deoxypyrimidine kinase activity.

The action of two restriction endonucleases on polyoma virus DNA has been examined and the sites at which they cleave the DNA located. One of the enzymes, KpnI from Klebsiella pneumoniae OK8, cleaves polyoma DNA twice at about 11.6 and 59.2% from the EcoRI site. The other enzyme, PstI from Providencia stuartii 164, cleaves polyoma DNA five times at about 14.8, 16.5, 32.6, 50.3 and 80.0% from the EcoRI site. Some of the cleavages produced by these enzymes alone, or in conjunction with other endonucleases, may be of use in the isolation of regions of particular interest from the virus DNA.

Infection of glucose, sulphur or nitrogen starved cells with MS2 virus results in the production of progeny virus but the absence of cell lysis and the failure of progeny virus release. Addition of glucose or sulphur to the correspondingly starved cells results in the normal release of virus within 40 to 60 min. Return of nitrogen to nitrogen-starved cells, however, does not result in the release of virus, even after 1 ½ h. In experiments with uninfected, starved cells it was found that glucose or sulphur starved cells begin dividing within 45 min after the limiting compound is returned. In contrast, nitrogen-starved cultures still have not begun to divide 1 ½ h after the return of nitrogen. The correlation between the time it takes for starved, infected cultures to resume lysis after the return of the limiting compound and the time similarly starved, but uninfected, cells normally begin division after addition of the limiting compound supports the hypothesis that lysis by RNA phage is related to cell division and may result at the time of cell division from failure of the cells to divide properly.

Primary human amnion cell monolayers which had been treated with DEAE-dextran, washed, and then inoculated with sonicated cells of the EB3 line of Burkitt's lymphoma cells developed foci of transformed amnion cells 7 to 14 days later. When either the DEAE-dextran or the sonicate was omitted, no significant transformation was found. The foci consisted of enlarging mounds of rapidly dividing cells, which upon subculturing continued their high mitotic activity; and strains or lines of the transformed amnion cells were thus readily established. The modal number of chromosomes in such lines was 65 instead of the normal 46. Not all human amnions yielded cells transformable by EB3 cell sonicate, as determined by direct comparisons using the same cultural conditions and testing with the same fresh sonicate preparation in the same experiment. Overall, it appeared that only about 40 to 50% of the amnions yielded transformable cell monolayers; the rest gave monolayers apparently completely refractory to the transformation. The transformed amnion cells contained nuclear and cytoplasmic Epstein-Barr virus (EBV) antigen(s), as revealed by indirect immunofluorescence tests.

EB3 cell sonicate also caused the appearance of rapidly growing transformed cell foci on secondary rat embryo cell monolayers which had been sensitized with DEAE-dextran.

Calcium in the cell maintenance medium decreased the number of transformed foci found, both on the human and on the rat cell monolayers.

Sonicates of cultured normal human leucocytes had no such transforming activity for either the human or the rat cells.

The transforming agent in EB3 cell sonicate was completely destructible by either deoxyribonuclease or trypsin, but not by ribonuclease, and was not neutralizable by anti-EBV serum. The simplest interpretation of these results is that the transforming agent is part or all of the EBV DNA plus some necessary protein, with both the DNA and the protein accessible to hydrolytic enzyme action.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-1 (17 syn +) have been characterized by SDS polyacrylamide gel electrophoresis.

Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells.

The different polypeptide patterns obtained at permissive (31 °C) and non-permissive (38 °C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA — ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles.

A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (1) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of the genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.

Protein synthesis in cucumber cotyledons infected with two strains of cucumber mosaic virus (CMV) differing in symptom type was investigated by the technique of radioactive double-labelling of polypeptides followed by analysis on SDS polyacrylamide gels. Radioactive labelling in the presence of actinomycin D revealed several virus-stimulated proteins associated with infection. In addition to coat protein, three polypeptides with apparent mol. wt. larger than that of coat protein, together with two smaller species were detected. No qualitative difference was observed in the patterns of protein stimulation which might account for the difference in symptoms induced by the two strains.

Serial passaging of Semliki Forest virus in BHK cells at a constant input multiplicity of 50 p.f.u./cell resulted in a 4 log10 drop in the yield of infectious virus by passage 9. An interference analysis showed that this drop was due to the presence of defective-interfering (DI) particles. Attempts were made to separate the DI particles from standard virus by equilibrium and velocity centrifugation. Only equilibrium centrifugation on CsCl resolved the DI particles (identified by interference analyses) from standard virus. The buoyant density of the DI particles (1.23 g/ml) was higher than that of standard virus (ρ = 1·20 g/ml).

No difference was observed between the structural proteins of standard virus and DI particles. Analysis of the RNA of standard virus and DI particles showed that whereas standard virus contained only 42S RNA (mol. wt. approx. 4·2 × 106), DI particles contained two smaller pieces of RNA of mol. wt. 0.81 and 0·75 × 106 respectively. Infectivity assays showed that these low mol. wt. species were not only non-infectious but also interfered with the infectivity of 42S RNA from standard virus. Nucleocapsids derived from purified DI particles had a buoyant density 0·02 g/ml greater than the nucleocapsids from standard virus. Analysis of the RNA from DI nucleocapsids showed it to be entirely of the low mol. wt. class.

To account therefore for the density difference not only between DI particles and standard virus but also between their respective nucleocapsids we propose that each SFV DI particle contains several molecules of the low mol. wt. RNA species.

Purified defective-interfering (DI) particles of Semliki Forest virus are unable to carry out any of the steps in virus multiplication except uncoating. Cells co-infected with DI particles and standard virus contain several virus-specified RNA species (DI particle-specific species) absent from cells infected with standard virus alone. Moreover, synthesis of all the virus-specified components distinctive of standard virus-infected cells is reduced. The DI particle-specific RNA species comprise two poly A-containing single-stranded RNAs (DIss1 and DIss2), identical to those found in purified DI particles, two double-stranded RNAs (RFs) and a new size class of replicative intermediate (RI).

Hybridization experiments showed that the nucleotide sequences of DIss1 and DIss2 (i) are present in the 42S genome of standard virus but absent from the 26S RNA — the RNA from standard virus-infected cells which encodes the structural proteins of the virion (Clegg & Kennedy, 1975a) and (ii) are complementary to the negative strands of the DI particle-specific RFs and RI. Oligonucleotide fingerprinting revealed extensive nucleotide sequence homology between DIss1 and DIss2. Analysis of the mRNA complement of standard virus-infected, co-infected and uninfected cells strongly indicated that neither DIss1 nor DIss2 can serve as a functional messenger RNA.

From these studies we propose a mechanism for the multiplication of and interference by DI particles of Semliki Forest virus.

A Pichinde persistently infected BHK21/13S culture was established in which defective interfering (DI) virus continued to be synthesized after cessation of plaque-forming virus replication. This DI virus, concentrated from NaCl-polyethylene glycol treated tissue culture fluids, was shown to band over a much broader range than standard virus, in either discontinuous or continuous sucrose gradients. The polyacrylamide gel profile of the RNAs extracted from standard virus contained six components with sedimentation coefficients corresponding to 31, 28, 22, 18, 15 and 4–6S. All RNAs extracted from DI virus preparations, however, did not contain the 22 and 15S species. Furthermore, a new 20S fraction was observed in DI virus taken from cultures which had been maintained for more than 175 generations after the initial infection, whereas it was absent in DI virus synthesized prior to that time.

The effect of interferon on the replication of vesicular stomatitis virus (VSV) and type-C oncornavirus in two Balb/c mouse cell lines, JLS-V5 and JLS-V9R, infected with MuLV-R was examined. VSV replication was inhibited threefold (0·5 log10) in both cell lines by 10 to 20 units of interferon/ml. In JLS-V5 cells C-type virus yields, as measured by 3H-uridine incorporation and reverse transcriptase activity, were also reduced three-fold by 10 to 20 units of interferon/ml. However, in JLS-V9R cells, C-type virus replication was refractory to interferon at concentrations up to 1 × 104 units/ml. Infectious C-type virus transmitted from JLS-V9R cells to Balb/3T3 cells was as sensitive to interferon as virus transmitted from JLS-V5 cells, indicating that resistance of C-type virus in JLS-V9R cells is a feature of the cells rather than of the virus strain.

The purified fibre and hexon of adenovirus 12 inhibit the transformation in tissue culture of murine sarcoma virus (MSV-M) by as much as 80% and 70%, respectively, when they are added to cells 8 to 20 h before MSV-M infection. During a 12 h period, only about 6 to 8% of added radiolabelled viral proteins become associated with cells (or 1·0 µg protein bound/105 cells). No inhibition occurs when the proteins are added simultaneously with MSV-M or 90 min or 4 h after MSV-M. There is also a direct correlation between the extent of focus inhibition and the concentrations of viral proteins used. Concentrations of viral proteins used to inhibit cell transformation do not affect cell growth, but do reduce cell macromolecular synthesis.

Using 5′-nucleotidase and NADPH:cytochrome c reductase as respective enzyme markers for the plasma membrane and endoplasmic reticulum, a satisfactory separation of these two membrane fractions from a cell line (SMB) derived from a scrapie mouse brain has been achieved. The coincident distribution of scrapie infectivity and 5′-nucleotidase in various fractions isolated from these cells indicates that most of the scrapie infectivity present in this cell line is associated with the plasma membrane.

Electron microscopic examination of poliovirus in CsCl-solutions without conventional staining with phosphotungstic acid or uranyl acetate revealed the existence of cores inside poliovirus particles. Empty capsids could not be visualized by this technique. Evidently, CsCl can enhance the contrast of the virus particle to an extent sufficient for the study of poliovirus particles by electron microscopy under conditions in which they exhibit specific properties.

A proteolytic activity has been found associated with purified preparations of sowthistle yellow vein virus. The activity selectively digests the M1 and M2 proteins after dissociation of the virus with Nonidet P 40 (NP40), and is not stopped by several inhibitors of proteases.

Chenopodium quinoa plants were inoculated with pairs of pseudo-recombinant isolates of raspberry ringspot virus that shared one part of their genome but not the other. Results of typing progeny virus obtained from systemically infected leaves indicated that RNA-1 from different virus strains differed in competitiveness, and also in ability to allow the expression of differences in competitiveness between RNA-2 from different strains. Ability of genotypes to dominate was related to the rapidity with which they induced systemic symptoms in singly infected plants.

An iridovirus, Apis iridescent virus (AIV), isolated from sick adult specimens of Apis cerana (Hymenoptera) from Kashmir, closely resembles iridescent viruses from Tipula and Sericesthis spp. (TIV and SIV). However, AIV is only distantly related serologically to TIV and SIV and is even more remotely related to several other similar viruses that were tested in tube precipitation tests with intact particles. AIV multiplied in Apis mellifera, forming cytoplasmic iridescent crystalline aggregates in several tissues, but unlike all the other iridoviruses tested, it failed to multiply in Galleria mellonella.

Introduction. The International Committee on the Taxonomy of Viruses (ICTV), which is a committee of the Section on Virology of the International Association of Microbiological Societies (IAMS), completed a round of meetings during the Third International Congress for Virology that was held in Madrid from 10 to 17 September 1975. Since ICTV only meets during these conferences, which are held every four years, the meetings are important occasions for reviewing the classification and nomenclature of viruses. Decisions on new names, which encapsulate the recognition of natural ‘groups’ of viruses, evolve slowly. Official approval for new names depends upon a series of sequential steps; recommendations by one or more of the subcommittees of the Executive Committee of ICTV (subcommittees on Bacterial, Invertebrate, Plant and Vertebrate Viruses respectively, and for some of the larger viral groups which span several kinds of hosts — the Coordination Subcommittee), which are considered by the Executive Committee of ICTV and may finally be submitted for approval by ICTV itself.