Epstein-Barr virus DNA synthesized in superinfected Raji cells.

Raji and P3HR-1 are established Burkitt lymphoma-derived cell lines that carry the Epstein-Barr virus (EBV) genome. Superinfection of the Raji cell line, a non-virus-producer, with virus from P3HR-1 cells results in the synthesis of several thousand copies of EBV-DNA per cell with attendant inhibition of synthesis and breakdown of Raji cell DNA. The DNA synthesized in superinfected Raji cells has been characterized. Raji cells infected with P3HR-1 virus and labeled with 32P 10 hr after infection synthesized only viral DNA. As much as 90% of the 55 S, 32P-labeled material was localized in the nucleus of superinfected cells after a 10-hr labeling period. The viral DNA purified from superinfected cells had the same buoyant density as the DNA isolated from P3HR-1 virions and after purification could be recovered with a specific activity exceeding 106 cpm/μg in an amount which approached 10 wg/107 infected Raji cells. The viral DNA from superinfected cells reassociated with the DNA from Raji or P3HR-1 cells and with the DNA from virus but did not reassociate with DNA from cell line 698 (a lymphoblastoid cell line lacking the EBV genome). Nuclei isolated from superinfected Raji cells incorporated label from deoxythymidine triphosphate into an acid-insoluble product, most of which had a buoyant density identical to that of DNA from P3HR-1 virions. Digestion of the DNA from superinfected cells with the restriction endonuclease EcoRI produced a number of fragments with molecular weights which ranged from less than 1 million to approximately 30 million when analyzed by electrophoresis on agarose gels. All of the fragments produced by digestion of DNA from virus were present in the digest of DNA from superinfected Raji cells.     

Morphologic heterogeneity in egg- and monolayer-propagated newcastle disease virus.

Newcastle disease virus (NDV) propagated in monolayers of chick embryo fibroblasts (M-NDV) has been found to differ morphologically from NDV propagated in embryonated eggs (E-NDV). Virions of M-NDV sediment faster, are lower in density, are about four times larger, and are more fragile to degradative forces encountered during high speed pelleting and rebanding in CsCl than E-NDV virions. These differences have been shown to be host controlled and are not due to contaminant virus, differences in growth medium, virion clumping, or time of harvest. Although both E- and M-NDV populations contain multiploid virions, these virions do not exhibit multihit inactivation kinetics. The possible genetic consequences of the differences between E-NDV and M-NDV are discussed.     

Defective interfering particles in monolayer-propagated Newcastle disease virus.

Newcastle disease virus (NDV) serially passaged in chick embryo fibroblasts (M-NDV) gives rise to defective interfering (NDV-DI) particles, while NDV passaged in embryonated eggs (E-NDV) does not. Co-infection with these particles and infectious virions results in a 99% reduction in yield. Interference is not due to interferon or to prevention of absorption of infectious virions and is specific for NDV. The particles mediating interference sediment at the same velocity as infectious virions. The accumulation of NDV-DI particles in monolayers but not in eggs may be a consequence of the fact that M-NDV virions are larger and probably contain more RNA, or it may reflect differences in NDV replicative processes in eggs and monolayers, or both.     

Identification of the human papovavirus T antigen and comparison with the simian virus 40 protein a.

The simian virus 40 (SV40) A protein (T antigen) and the early proteins produced by cells infected with BKV-type human papovaviruses have been compared by immunoprecipitation and tryptic peptide analyses. Cells infected with the human viruses and transformed cell lines synthesize phosphorylated proteins that interact with antiserum against the SV40 T antigen. These proteins cannot be distinguished from the SV40 A protein by sodium dodecyl sulfate-gel electrophoresis. Peptide map analyses have shown that the human virus proteins are similar, but not identical, to the SV40 A protein. Because of the distinct, reproducible differences in the peptides of the papovavirus proteins, structural analyses provide a direct procedure for detecting the expression of each type of virus in cells.     

Mechanism of infection by Epstein-Barr virus. II. Comparison of viral DNA from HR-1 and superinfected Raji cells by restriction enzymes.

Virus DNA (RS virus DNA) was directly isolated from Raji cells superinfected with Epstein-Barr virus derived from P3HR-1 cells and compared with original superinfecting virus DNA from P3HR-1 cells (HR-1 virus DNA) in agarose-gel electrophoresis after digestion with various restriction enzymes. EcoR-1 digestion of RS virus DNA produced 15 fragments identical to those from HR-1 virus DNA. However, two fragments, EcoR1 No. 6 (10 × 10⁶ daltons) and EcoR1 No. 11 (4.6 × 10⁶ daltons), observed in HR-1 virus DNA were not detected in RS virus DNA from superinfected Raji cells. In addition, the EcoR1 No. 4 (13.5 × 10⁶ daltons) fragment of RS virus DNA showed a molar ratio of 2 whereas HR-1 virus DNA produced the same fragment with a molar ratio of 1. Electrophoresis patterns of virus DNA digested with Hind III, Bam H-I, Hpa I, and Sal I were also examined. In general, both types of virus DNA produced similar patterns after gel electrophoresis, with minor differences in molar ratios after being treated with the restriction enzymes suggesting that RS virus DNA obtained by superinfection of Raji cells is basically identical to HR-1 virus DNA but may contain a population of DNA a little more heterogenous than HR-1 virus DNA.     

Phage lambda DNA injection into Escherichia coli pel- mutants is restored by mutations in phage genes V or H.

Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R_f and a 0.5 R_f activity. The 0.2 R_f enzyme was found only in actively growing cells, while the 0.5 R_f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R_f form of this enzyme.SV40 wild-type and SV40 tsBC and tsC mutants stimulated the levels of the 0.2 R_f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 tsA mutants (tsA7, 28, 30, 58, and 209) and two different SV40 tsD mutants (tsD202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R_f thymidine kinase activity could be detected in tsA or tsD mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R_f thymidine kinase activity at both permissive and nonpermissive temperatures.These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.     

Some properties of carnation ringspot virus single- and double-stranded ribonucleic acid

From sodium dodecyl sulfate (SDS)-dissociated carnation ringspot virus (CRSV) two RNAs of different sizes were separated by density gradient centrifugation. Preparations of the smaller RNA-2 (0.5 × 10⁶ daltons) were not infectious; preparations of the larger RNA-1 (1.5 × 10⁶ daltons) were only moderately infectious. Mixtures of the two kinds of preparations were very infective. This enhancement of infectivity was demonstrated within and between the two RNAs from each of two strains of CRSV. The genetic information controlling irreversible virus particle aggregation and dissociation of virus particles by SDS at pH 5 was contained in RNA-1. Virus-infected tissue contained dsRNA replicative forms corresponding to RNA-1, RNA-2, and a minor ss-RNA component.     

Parental G protein reincorporation by a vesicular stomatitis virus temperature-sensitive mutant of complementation group V at nonpermissive temperature.

Rescue virions obtained after superinfection of ts O45(V) Indiana serotype-infected cells by uv-irradiated vesicular stomatitis virus (VSV) at a nonpermissive temperature do not incorporate G protein synthesized at this temperature. They apparently contain G protein in their envelope since they are neutralized by homotypic antivirion and antispike sera. Rescue of ts O45(V) mutant Indiana serotype by uv-irradiated VSV New Jersey serotype is demonstrated. Ultraviolet-inactivated VSV of New Jersey serotype has therefore been used to determine the origin of the incorporated G protein molecules. The progeny-rescue virions belong genetically to VSV Indiana complementation group V. They are however neutralized by anti-New Jersey serum and not by anti-Indiana serum. Rescue ts O45(V) virions have thus probably reincorporated G protein molecules supplied by the uv-irradiated New Jersey virus. This provides further evidence suggesting that protein G is encoded for by gene V.     

Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

Parental and replicating vaccinia DNA molecules, labeled with [³H]thymidine or [¹⁴C]thymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90–92 S) were “nicked”; parental DNA molecules sedimenting at 70–72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90–92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2–3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-1 nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes.     

Radiochemical identification of the kil gene product of bacteriophage lambda.

We have identified the coliphage λ kil gene product using a differential labeling technique. The kil gene polypeptide has a molecular weight of about 16,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of the kil protein indicates that it may exist as a tetramer in native form.     

Studies on polylysogens containing lambda N-cI- prophages. II. Role of high multiplicities in lysogen formation by lambda N-cI- phage

Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities. The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper. The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i. of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i. as low as 12. All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.     

Size distribution and In vitro translation of the RNAs isolated from turnip yellow mosaic virus nucleoproteins

Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology12, 521–539, 1960). When analysed by polyacrylamide-gel eletrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 × 10⁶ daltons) was present only in nucleoproteins B₁ and B₂. Nucleoproteins B₀, B₀₀, and B₀₀₀ contained RNAs ranging in size from approx 1.3 to 0.28 × 10⁶ daltons. The 0.28 × 10⁶-dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B₀₀₀ and B₀₀. Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 × 10⁶-dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated.     

Biological properties of the VSV glycoprotein. II. Effects of the host cell and of the glycoprotein carbohydrate composition on hemagglutination.

Virions of the Indiana serotype of vesicular stomatitis virus (VSV) grown in HKCC, BHK21-F, and MDBK cells have been found to differ considerably in their ability to agglutinate goose red blood cells and in their pH optimum for hemagglutination. Virions grown in HKCC cells had a specific hemagglutinating activity approximately 20 times greater than those grown in BHK21-F cells, and approximately 40 times greater than those grown in MDBK cells. The differences in activity and pH optima for hemagglutination were also found with the glycoprotein isolated from virions grown in the different cells. Carbohydrate analyses of the glycoproteins isolated from virions grown in the different cells revealed differences in carbohydrate composition. The observed differences in total carbohydrate and sialic acid content of the viral glycoproteins, and their correlation with differences in hemagglutinating activity provide additional evidence for a role of carbohydrate in virus-receptor interactions and suggest a chemical basis for the host-cell modification of the hemagglutinating activity of VSV.     

Circular monomers of bacteriophage lambda DNA as substrates for in vitro packaging

Monomeric circular λ DNA molecules were isolated from phage-infected cells, purified, and their biological activity tested using in vitro DNA packaging and λ terminase assays. These circles, which contain a single cos, proved to be as efficient as linear λ concatemers both for packaging into viable phage and cleavage at cos by highly purified λ terminase.     

Replication of Theiler's murine encephalomyelitis viruses in BHK21 cells: an electron microscopic study

The replication of two different Theiler's murine encephalomyelitis viruses (GDVII and DA) in BHK21 cells was studied by electron microscopy. During the early stages of infection, the changes observed in infected cells were identical for both viruses, and typical of those described in the literature for other picornaviruses. However, fundamental differences in the intracellular development of GDVII and DA viruses were observed late in the infection. Well-developed crystalline arrays of virions were observed in the cytoplasm of GDVII virus-infected cells, and virus was readily released upon cell lysis. In contrast, DA virus did not form similar crystals. Instead, in DA virus-infected cells unique membranous structures consisting of two membrane units enclosing a layer of virions one particle deep were regularly observed in the cytoplasm at late times in the infection. In addition, DA virus did not appear to be freely released upon cell lysis. Single cell growth kinetics of GDVII and DA viruses in BHK21 cells showed a close correlation with the electron microscopic observations. While GDVII virus was released from cells DA virus remained cell associated. The proliferation of similar membranous structures in parallel with maturation of virus particles has not been described previously in electron microscopic studies of other picornaviruses.     

The induction of a mutant prophage lambda in Escherichia coli: a rapid screening test for carcinogens.

The induction of prophage λ and its mutant, λcIts857, by a number of water-soluble substances, carcinogens and noncarcinogens, has been investigated with the object of developing a suitable screening test for carcinogens and of assessing the sensitivity of repressors to inducing agents. By the use of measurements of endolysin activity in place of plaque assays, induction may be estimated more simply and quickly. While prophage λ is inducible only by certain carcinogens, λcIts857 is inducible, without exceptions, by all the carcinogens tested. Some carcinogens require prior metabolic activation to be effective. Most noncarcinogens are noninducing toward both strains. The prophage λcIts857 induction test appears to be a simple, quick, and sensitive method for the screening of carcinogens. It can detect carcinogens that do not induce the wild-type prophage λ, and, in some cases, is even more sensitive than the Ames Salmonella/microsome test.     

Isolation and mapping of Mu nu mutants which grow in him mutants of E. coli

Mu nuA and Mu nuB mutants were selected by their ability to form plaques on lawns of Escherichia coli himA and himB mutants, respectively. Deletion mapping of the nuA and nuB mutations by marker rescue from λpMu transducing phages or from hosts containing deleted Mu prophages indicated that nuA mutations are located within or to the left of gene A and that nuB mutations are located within or between Mu genes G and I.     

DNA packaging in the lambdoid phages: the role of lambda genes Nu1 and A

Of all the morphogenic genes of the coliphage λ, only Nu1 and A are both necessary and sufficient for terminase activity in vitro. Previously we have reported that extracts of Nu1^?- and Aam^?-infected cells will not complement each other to promote packaging in an in vitro assay (A. Becker, H. Murialdo, and M. Gold, Virology, 78, 277–290, 1977). We report here that such complementation is possible under certain conditions; furthermore, complementation appears to proceed in a noncatalytic fashion. The product of gene Nu1 (gpNu1) therefore does not appear to be involved in the synthesis of the product of gene A (gpA). We discuss the hypothesis that λ terminase is a heterodimer consisting of gpNu1 and gpA subunits, and show that neither of these subunits alone is able to promote either the DNA-cutting or prohead-filling events of packaging.