- Volume 49, Issue 1, 1980

The replication of bacteriophage φX-174 is impaired at temperatures above 40 °C. Mutants (ht) that replicate at high temperature were isolated and partially characterized. Wild-type φX fails to grow at high temperature because, unlike the mutants, it does not make appreciable amounts of single-stranded (ss)DNA. An unusual form of ssDNA, not found in complete virions, is described.

The double-stranded (ds)DNAs of the three closely related temperate Pseudomonas pseudoflava bacteriophages gd, ge and gf were studied biochemically and biophysically. The GC content of the DNA was 67.4 ± 0.5% and differed only slightly from that of the host P. pseudoflava. By electron microscopic length measurements a mol. wt. of 26.1 × 106 to 26.7 × 106 was calculated for the three bacteriophage DNAs. Homogeneity of the bacteriophage DNAs was further demonstrated by specific cleavage with restriction endonucleases EcoRI and HindIII. It was concluded that the three homo-immune bacteriophages are identical. The genome size of the host P. pseudoflava GA3 was 3.7 × 109 as calculated from optical renaturation rate measurements with Xanthomonas pelargonii reference DNA. The bacteriophage gd genome thus amounts to 0.7% of the chromosome of this bacterial host.

The delay in the replication of herpes simplex virus surviving u.v. irradiation occurs after the uncoating of virus, as judged by sensitivity to DNase. It occurs before translation, judged by the kinetics of appearance of various virus-specific proteins, and before transcription, judged by the detection of virus-specific RNA by in situ hybridization. Since the delays in both transcription and translation are reversed by photoreactivation, the simplest hypothesis is that pyrimidine dimers directly obstruct transcription; unless these are broken by photoreactivating enzymes, there will be transcriptional delay until reactivating processes have repaired the lesion. The u.v. sensitivities of the abilities to induce various enzymes (thymidine kinase, DNase and DNA polymerase) were only about four times less than that of infectivity. The ability to induce the three enzymes was three times less sensitive than that of the structural antigen (Band II).

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

The ultrastructure of the small intestine of gnotobiotic lambs infected with lamb astrovirus was studied. The virus was observed from 14 to 38 h p.i. in mature columnar epithelial cells covering the apical two-thirds of villi. Crystalline arrays of virus particles with a centre to centre distance of approx. 29 nm were seen in the cytoplasm and virus particles were also observed in apical pits and tubules and in lysosomes. Macrophages containing virus particles in lysosome-like organelles were seen in the lamina propria. Virus particles were released by desquamated cells disintegrating in the gut lumen. Cuboidal cells lining villi appeared from 38 to 70 h p.i., and by 120 h p.i. the villi appeared normal.

The antigenic relationship between human coronavirus strain 229E (HCV 229E) and mouse hepatitis virus strain 3 (MHV 3) was studied by means of the indirect form of enzyme-linked immunosorbent assay (ELISA). A cross-reaction was found with hyperimmune rabbit sera between HCV 229E and MHV 3 which may be due to the adherence of bovine serum components from tissue culture media, which were present on virus particles even after extensive purification. No cross-reaction was observed with immune sera absorbed with bovine serum, or with HCV 229E grown in tissue culture without serum. This indirect ELISA with HCV 229E may prove to be useful for studies with human sera.

Conditions are described for the production of 0.3 to 0.7 NIH mouse reference standard units of interferon per cell from Ehrlich ascites tumour cells cultured as monolayers and induced by infection with Newcastle disease virus (NDV). Inclusion of theophylline (6 mm) in the medium increased the interferon yield three to four times. Cells infected with NDV started to lyse at about 15 h p.i., but infected, theophylline-treated cells lysed only 24 h p.i.

Several other methylxanthines (e.g. theobromine, caffeine and isobutylmethylxanthine), when tested at concentrations similar to that of theophylline, did not boost interferon production. Dibutyryl cyclic AMP (10−6 to 10−2 m) did not substitute for theophylline in increasing interferon production, and, if used together with theophylline, did not cause further enhancement.

Seventeen temperature-sensitive mutants of equine arteritis virus, a nonarthropod-borne togavirus, have been isolated. 5-Fluorouracil, 0-methylhydroxylamine and ethyl methanesulphonate were used as mutagens. The mutants were characterized by their ability to synthesize virus RNA and virus proteins at the permissive (35 °C) and restrictive temperature (40 °C) using autoradiography of cells labelled with 3H-uridine in the presence of actinomycin D and immunofluorescence respectively. Among the mutants, four were unable to synthesize virus RNA and virus proteins at 40 °C (RNA−/protein−). The other mutants were RNA−/protein+ (3); RNA±/protein− (2); RNA+/protein+ (6) and RNA+/protein− (1).

Starting with cloned NIH 3T3 mouse cells we have isolated a series of related lines infected with the Kirsten murine sarcoma/leukaemia (MSV/MLV) virus complex. These lines exhibit all three possible infected cell phenotopes: (i) transformed MSV/MLV producers; (ii) non-transformed MLV producers; (iii) transformed non-producers. We have also selected non-transformed revertants from one of the non-producer clones. This series of lines allows the study of the expression of the virus genome against a constant background of cellular gene expression. We have further characterized the lines with regard to anchorage dependence of growth, tumorigenicity and the presence of a rescuable sarcoma genome. The non-producer clones are uniform in their transformed properties. The revertants contain rescuable sarcoma virus, biologically indistinguishable from the original transforming virus, implying that the reversion is due to a change in cellular rather than virus genetic information.

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK− viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

Treatment of tick-borne encephalitis (TBE) virus with Triton X-100 (TX-100), octylglucoside (OG) or cetyltrimethylammonium bromide (CTAB) caused dissociation of the virus envelope into dimers or monomers of the glycoprotein V3. By centrifugation into detergent-free sucrose density gradients, these subunits were found to reassociate and to form haemagglutinating homogeneous glycoprotein complexes sedimenting at 15 to 16, 16 to 18 and 11 to 12S after TX-100, OG and CTAB treatment, respectively. Glycoprotein complexes obtained after TX-100 solubilization contained less than 1% lipid and detergent by weight.

The ts lesion of the fowl plague virus (FPV) mutants ts 18 and ts 236 has been located in RNA segment 2 (Ptra gene, corresponding to P3). After double-infection with these mutants and ts 90 or ts 93, which also carry a ts lesion in segment 2, plaques were formed at the non-permissive temperature (40 °C). These plaques cannot be passaged at 40 °C and exhibit a morphology which differs from those formed by the wild-type virus. The yield of infectious particles after double-infection shows a non-linear correlation between the plaque number and dilution, indicating that at least two particles are needed for infection of a cell. All experimental evidence points to an intracistronic complementation within the P3 protein.

Four mutants of Semliki Forest virus (SFV) strain L10 showing altered virulence in weanling mice were selected following mutagenesis with N-nitro-N-methyl-N′-nitrosoguanidine. Intraperitoneal (i.p.) injection of wild-type (wt) virus resulted in the death of all mice given more than 30 p.f.u. Protection of mice surviving injection with lower doses of virus was not obtained. A proportion of mice infected with doses from 104 to 107 p.f.u. of mutant virus survived infection. Such surviving mice were protected against an otherwise lethal challenge with wt virus. Three mutants which were non-lethal at doses of 102 p.f.u. were studied further. The mutants M4 and M103 established a transient viraemia which was quickly cleared; unlike the wild-type, the virus did not enter the brain or spinal cord. The mutant M136 produced a viraemia similar to the wild-type, but reached a lower titre in the brain; it was not recovered from the spinal cord. Severe necrotic and inflammatory lesions, associated with large numbers of virus particles, were present in the brain and spinal cord (CNS) of mice 5 days p.i. with wt virus. Mice infected with M136 showed degenerative lesions in the grey and white matter of the CNS at 13 days p.i. Virus particles could not be detected at this time either by electron microscopy or infectivity assays.

Thirteen temperature-sensitive (ts) mutants of HSV-1 were analysed for their capacity to establish latent infections in the brains of mice. Eleven of the mutants could be classified as latency-positive or -negative; two could not be assigned to either group. Leakiness of mutants in the brain and differences in particle/infectivity ratios were found not to play a role in the results. Ts + revertants of selected latency-negative mutants regained the capacity to establish latent infections, indicating that it was the ts lesion in these agents which was involved in latency. Ultrastructural studies of neuroblastoma cells infected with various mutants and maintained at the restrictive temperature showed that no absolute correlations could be made between capacity to establish latent infection and synthesis of various morphologically identifiable virus products. Finally, from a comparison of latency characteristics with previously established polypeptide phenotypes of the mutants it was concluded that one immediate early and one or more later virus functions are necessary for establishment and/or maintenance of the latent state.

Purified preparations of the CVS strain of rabies virus, which were labelled during the infectious growth cycle with different isotopes or labelled in vitro by iodination or acetylation, contained five major proteins, L, G, N, M1 and M2, when examined by polyacrylamide gel electrophoresis (PAGE). The major surface glycoprotein, G, could be separated into two components, G1 and G2, in some PAGE systems; they were present in about equimolar amounts and had apparent mol. wt. of 70.5 × 103 and 65 × 103, respectively. The virus nucleocapsid (ρ = 1.28 g/ml) could be isolated after detergent treatment of purified virus. It contained the virus RNA, the major nucleocapsid protein, N (mol. wt. 58.5 × 103), and small amounts of a large protein, L (mol. wt. 170 × 103). Two membrane proteins, M1 (mol. wt. 39.5 × 103) and M2 (mol. wt. 25 × 103), were also observed. Chromatography of dissociated rabies virus in agarose columns with guanidine hydrochloride did not resolve any additional virus structural proteins. Two-dimensional peptide map analysis of iodinated structural proteins indicated that they were unique gene products and not derived from a precursor polypeptide by cleavage. The peptide maps of the two glycoproteins, G1 and G2, appeared identical. The approximate number of protein molecules per virion has been determined. Rabies virus-directed protein synthesis in BHK21 cultures was detected as early as 6 h p.i. and all the proteins were present 12 h p.i. Additional non-structural virus-specific proteins were not observed. The NaCl hypertonic shock procedure, which differentially inhibits polypeptide chain initiation in different classes of mRNAs, was used to inhibit the synthesis of the G and M1 proteins relative to the others selectively. All the rabies virus proteins were synthesized simultaneously following release from hypertonic treatment, suggesting that there are independent polypeptide chain initiation sites for the synthesis of each of the rabies proteins and that each protein is derived via translation of monocistronic mRNA species.

Rabies virus polysomes contained two sizes of messenger RNAs, one of which had a sedimentation value of 30S and another which sedimented at 12 to 16S. RNA extracted from infected cultures contained virion-size RNA, 42S, as well as 30S and 12 to 16S RNA species. Hybridization studies indicated that the 30S and 12 to 16S RNAs had nucleotide sequences which were complementary to virion RNA. RNA isolated from virus polysomes contained adenylate-rich sequences which were heterogeneous in size and were determined to be about 100 to 250 nucleotides in length on the basis of their migration rates in polyacrylamide gels. Acid-urea agarose gel electrophoresis established that the 30S RNA material was composed of a single RNA species (mol. wt. ⩾ 1.65 × 106), whereas the 12 to 16S material could be resolved into at least four distinct species whose mol. wt. ranged from 0.28 to 0.87 × 106. When labelled rabies-infected cell RNAs, which were purified by oligo(dT)-cellulose chromatography, were annealed to excess unlabelled virus RNA, digested with ribonuclease T2 and the RNA duplex molecules analysed by polyacrylamide gel electrophoresis, five duplexes could be separated. The mol. wt. of these duplexes were estimated to be 3.2, 1.4, 0.96, 0.55 and 0.39 × 106, when compared to the known mol. wt. of vesicular stomatitis virus (VSV) RNA duplexes. This study suggests that the replicative processes of rabies virus are very similar to VSV and that rabies virus proteins are probably translated from smaller than virion-size RNAs.

By filtration of normal human serum through a Sephadex G-200 column, an anti-virus release factor (AVRF), which is capable of inhibiting the release of Epstein-Barr virus (EBV) from cells cultured in vitro, was found in the fractions corresponding to IgM. Another component, antagonistic to the activity of AVRF, was found in the fractions close to those of albumin. Both AVRF and anti-AVRF were found in all sera from four EBV seropositive and three seronegative adults tested. EBV-release inhibition by AVRF was reversible. AVRF did not neutralize virus infectivity or inhibit intracellular virus growth. Virus adsorption on to cells was not prevented by AVRF but cap formation of EBV antigens on cells was augmented by it.

The ability of spleen macrophages to support Friend leukaemia virus replication was studied by testing for infectivity in adherent cells obtained from mice at various times after infection. Virus-releasing macrophages appeared early after infection and reached a high proportion. The released virus was probably synthesized de novo since macrophage infectivity was strongly reduced by treatments which affected cellular viability or synthetic activities and electron microscopy showed C-type particles budding from macrophages.