- Volume 24, Issue 2, 1974

Antibodies induced by the injection of alfalfa mosaic virus (AMV) or the protein of turnip crinkle virus (TCV) reacted with the viruses and their respective protein subunits in gel-diffusion tests. The ratios of virus-reactive antibodies to protein-reactive antibodies were different in two fractions obtained by chromatography of the sera on DEAE-Sephadex. The first DEAE-Sephadex fraction contained a greater proportion of virus-reactive antibodies than did the second fraction or the unfractionated antiserum. Electrophoretic mobility measurements at pH 8.6 showed that AMV and TCV had a greater negative charge per unit surface area than did their respective proteins.

Abortive transformation of human embryonic fibroblasts was induced by Molluscum contagiosum virus. The parameters studied in order to test the establishment of a persistent infection and transformation of cells were: (1) virus interference and interferon production; (2) alterations of cell morphology and growth; (3) presence of neoantigens; (4) karyotype. The possible mechanisms leading to an abortive transformation are discussed.

Cells infected with temperature-sensitive mutants of adenovirus type 5 have been labelled with [35S]-methionine and extracts examined by polyacrylamide gel electrophoresis followed by autoradiography. With the exception of one group of mutants, cells pulse-labelled for short periods (< 1 h) showed similar patterns of polypeptide synthesis whether they were infected with wild-type virus or with the mutants. However, on longer labelling it was evident that certain polypeptides were relatively less well labelled in the cells infected with the mutants at the restrictive temperature. A pulse-chase experiment suggested that preferential degradation of these polypeptides was occurring presumably by a proteolytic surveillance mechanism which could recognize defective polypeptides. One group of mutants showed no synthesis of capsid polypeptides at the restrictive temperature, in agreement with the results of previous serological investigations.

The effect of superinfection of adenovirus-infected cells with poliovirus in the presence of guanidine (P+G) has been analysed by pulse-labelling of cells with [35S]-methionine followed by polyacrylamide gel electrophoresis and autoradiography. The addition of P+G while reducing total polypeptide synthesis selectively inhibits cellular polypeptide synthesis and allows the visualization, particularly early in infection, of other labelled polypeptides previously obscured. Thus, at least three polypeptides i.e. ICSP-3, core-1 and ‘p-core 2’ can be detected in the absence of virus DNA synthesis. The addition of P+G also causes a retardation of the synthesis of the virus-induced polypeptides giving a clear sequence of translation viz ICSP-3: core-1: p-core 2 → ICSP-1 → hexon → penton base → fibre. Since a similar sequence can be demonstrated by the use of the inhibitor cordycepin, it seems possible that this reflects a sequence of transcription. P+G addition also effectively inhibits the formation of virus particles.

The principal cytosol thymidine kinase activity of African green monkey kidney cells increased approximately threefold at 21 to 29 h after infection with adenovirus type 5. Disc polyacrylamide gel electrophoresis (disc PAGE) analyses showed that the electrophoretic mobility relative to the tracking dye (Rm) of the cytosol thymidine kinase from both mock-infected and adenovirus type 5-infected cells was about 0.23. The cytosol thymidine kinase from normal cells also resembled the cytosol enzyme from infected cells with respect to phosphate donor specificity and sedimentation coefficient. Mitochondria from normal and virus-infected cells contained a cytosol-like enzyme and, in addition, a distinctive mitochondrial isozyme exhibiting an Rm of about 0.6 and a smaller sedimentation coefficient than the cytosol enzyme. The activity of the mitochondrial-specific isozyme of thymidine kinase (Rm = 0.6) was not significantly increased by virus infection. The ratio of the two thymidine kinase activities found in mitochondria also was not markedly changed by virus infection. The results suggest that adenovirus type 5 infection reactivates an inactive molecular form of cytosol thymidine kinase or derepresses the synthesis of the cytosol enzyme, but not that of the mitochondrial-specific thymidine kinase. Adenovirus infection does not alter the electrophoretic mobilities of the cytosol and mitochondrial thymidine kinases.

Two continuous cell lines derived from suckling mouse brain cells chronically infected with Japanese encephalitis virus were studied. One cell line underwent 122 passages, and the other 35 passages. Seven single-cell clones were obtained from the first culture. Both cultures released virus into the culture medium that differed from the initial virus in resistance to ribonuclease and urea, that had no haemagglutination activity and was not neutralized by the virus-specific serum. These properties are considered phenotypic changes in as far as intracerebral passages in mice or in competent cell line restored corresponding markers of the initial virus. The results obtained are compared with other chronically virus-infected cell cultures.

Rapidly reannealing DNA, containing repeated base sequences, was shown to be present in the genomes of four Rhizobium bacteriophages by studies of renaturation kinetics. Estimations of the mol. wt. of the DNA by measurement of kinetic complexity gave values of 1.3 × 108 for phage c, 1.6 × 108 for phage I, 9.4 × 107 for phage e and 8.2 × 107 for phage a. Comparison of the phage DNAs by annealing studies confirmed the classification of the phages into two groups.

Purified Penicillium stoloniferum viruses PsV-F and PsV-S both possess virus-associated RNA polymerase activity. The PsV-F polymerase was dependent upon GTP, ATP and magnesium chloride. It was not activated by Triton X-100. No DNA synthesis was observed when deoxyribonucleoside triphosphates were substituted for RNA precursors. The RNA reaction product for both viruses remained associated with the virus in sedimentation and electrophoresis and was resistant to digestion by pancreatic ribonuclease both before and after extraction from the virus. Extracted PsV-F RNA reaction product co-electrophoresed with parental double-stranded RNA.

The treatment of vesicular stomatitis virus with 1% saponin induced the release of envelopes from inner virus structures. The density of treated virus particles in CsCl increased from 1.22 to 1.23 g/ml, probably owing to loss of lipids. The envelopes could be removed from the nucleoprotein helix and further purified by repeatedly ultracentrifuging in sucrose gradients. The haemagglutinating activity of the envelopes was retained. The purified envelopes had sedimentation coefficients of 380 to 400S and a density of 1.20 g/ml. Two proteins, glycoprotein G and large protein L, were dominant in polyacrylamide gel electrophoresis of purified envelopes. In electron microscopy small amounts of ribonucleoprotein N were also seen as free, unwound ribbons, close to the envelopes.

Vesicular stomatitis virus (VSV) can form ‘pseudotype’ particles with neutralization antigen(s) derived from mouse or chicken leukaemia viruses. These pseudotype particles contain the genome of VSV, but show neutralization, host-range and interference specificity of the corresponding oncornavirus. Pseudotypes were also detected following the growth of VSV in two established cell lines derived from human tumours: in MaTu derived from mammary carcinoma and in Tu-135 derived from a sarcoma. No virus-like particles were detected in these cells by labelling with [3H]-uridine followed by sucrose density gradient sedimentation. Media from these cultures did not contain DNA polymerase. Nevertheless, the capacity to produce VSV pseudotype was transmitted to some pseudotype-negative cells following their co-cultivation with X-irradiated, pseudotype-positive cells. Cell-free filtrates did not transmit the property. An antigen detected by immunofluorescence was also consistently transmitted. The VSV pseudotype produced in cells infected by co-cultivation possessed the same neutralization specificity as VSV pseudotype produced in the original tumour cells.

DNA extracted from frog virus 3 has a GC mol fraction of about 0.56. The mol. wt. of the DNA is about 100 × 106 (97 × 106 by neutral sucrose gradient sedimentation; 102 × 106 from renaturation kinetics). Analysis by neutral sucrose sedimentation indicated that the DNA exists as a single heteroduplex in the virus particle. Alkaline sucrose gradient sedimentation indicated that single strand alkali labile interruptions probably occur in both strands of the heteroduplex. Renaturation kinetic analysis also indicated that about 7% of the genome contains repeated sequences. Quantitative analyses of DNA—DNA homology showed no sequence homology between frog virus 3 DNA and the DNA extracted from iridescent virus types 2, 6 or 9. This lack of sequence homology reflects the markedly distinct profiles on acrylamide gels of the structural polypeptides of frog virus 3 and the iridescent viruses.

A double lysogenic Bacillus cereus strain w (β, wx) induced with mitomycin C liberates phages β and wx. In addition the lysis of the organism is associated with the accumulation of a bacteriocin which was identified as phospholipase A. When the bacteria were cured of prophage wx they lost the enzyme production ability; the w (β) bacteria, named for convenience as cin − bacteria, could be converted to phospholipase A production by wx phage infection. Phage wx and its mutant wxc were very slowly adsorbed by cin − bacteria in a liquid medium and did not propagate noticeably, but the presence of agar led to normal multiplication. The rate of lysogenic conversion was also enhanced by agar. The bulk of the enzyme produced by the convertant was isolated as a homomolecular protein and identified as phospholipase A.

Characteristic ‘two-side’ electron microscope images and three-dimensional image reconstructions confirm that the surface structure of polyoma virus is based on the icosahedral T = 7 lattice. Tilting experiments show that the lattice is right handed (T = 7d).

Dose-response studies were performed in relation to the rate of uptake of [3H]-2-deoxyglucose by murine sarcoma virus-infected murine cells. A linear relationship was observed between the virus dilution used for infection and the rate of dgl uptake by the infected cells. The extrapolated dilution factor (reciprocal of dilution) at which no difference was observed between infected and uninfected cells was directly proportional to the activity estimated by focus assay of MSV. Such end-point dilution factors can be reproducibly and objectively obtained 2 to 4 days after infection.

Cell fusion of Ehrlich ascites tumour cells caused by antigen-antibody complexes of Sendai virus was studied by electron microscopy. The fusion process was similar to that produced by virus. The fusion of envelopes of virus particles aggregated by antibodies and cell membranes was not observed under the conditions employed, though cell fusion was enhanced. This finding supported the hypothesis that the formation of cell-cell bridges but not of cell-envelope-cell bridges is involved in the initiation of cell fusion.

Complexes consisting of only Dane particles are found frequently in Hepatitis B antigen-positive sera. It is proposed that the reason for their presence is the existence of a third antigen-antibody system involving the Dane coat material but not the small spheres or tubules.

Caffeine reduced the survival of u.v.-irradiated herpes simplex type 1 virus. The relative reduction of plaque count by caffeine was most pronounced in the second component of the survival curve. With more heavily irradiated virus, when multiplicity reactivation apparently played a major role, the caffeine effect was less marked.

Single-stranded polynucleotides (poly I and poly C), which do not generally have antiviral activity, became effective inducers of interferon when aggregated with different polycations. Poly I was consistently more active than poly C. The biological activity of the complexes depended more on the nature of the polycationic component than on the presence of the complementary base residue: introduction of covalently bound cytosine residue into the poly I—polybase complex did not have any significant effect.

The early stages of infection of Chenopodium amaranticolor leaves with two temperature-sensitive mutants of tobacco rattle virus and the wild-type strain from which they were derived were investigated by following the development of resistance of potential lesions to inactivation by u.v. light. Resistance began to increase between and 3 h after inoculation under permissive conditions (wild-type strain at 20 °C or 30 °C, mutants at 20 °C), but failed to occur with the mutants under restrictive conditions (30 °C). These results imply that at 30 °C the RNA of the mutants fails to replicate in the initially infected cells.