- Volume 47, Issue 1, 1980

Two forms of DNA may be isolated from gently lysed T5-infected bacteria. In neutral sucrose gradients a fast sedimenting form (fsf) is distinguishable from a slow sedimenting form (ssf) which moves at a rate similar to that of DNA extracted from mature T5 virus particles. A method of agarose gel electrophoresis is described which gives complete separation of the slow sedimenting from the fast sedimenting form. The DNA was extracted from the agarose using potassium iodide and isopycnic centrifugation; samples of the slow sedimenting form prepared by these methods were suitable for detailed structural studies.

The intracellular DNA of bacteriophage T5 contains two major DNA forms distinguishable by sedimentation rate through neutral sucrose gradients. The slow sedimenting form (ssf) which moves at a rate similar to that of DNA extracted from mature T5 virus particles, was prepared free of the fast sedimenting form and capsid-associated DNA by electrophoresis on agarose gels in which it migrated as peak II DNA. The ssf DNA thus obtained was subjected to extensive structural analysis. The number and location of single-stranded regions was studied using the single-strand-specific S1 nuclease and electron microscopy. The frequency and position of the single-stranded regions, which could occur both at internal and terminal locations, was reproducible in the population of ssf DNA molecules as a whole but individual molecules were not identical. After analysis of these results in conjunction with results of studies on the location of single-strand interruptions in ssf DNA it is suggested that single-stranded regions mainly occur at those sites corresponding to the sites of the major nicks in mature T5 virion DNA. When the ssf was isolated by gel electrophoresis and maintained in low ionic strength solutions it was found to be associated with several phage-specific proteins including some which are constituents of the mature virus particle. The presence of these proteins reduced the electrophoretic mobility of the DNA from that expected on the basis of its mol. wt. alone. This property of reduced mobility could be reproduced in vitro using exogenous ssf DNA and crude extracts from phage-infected, but not from uninfected, cells. By contrast, when DNA purified from phage T7 particles was incubated with crude extracts of T5-infected bacteria, the electrophoretic mobility of the T7 DNA was unaltered; thus the effect was due to T5-specific proteins on T5-specific DNA.

The contour lengths of both peak II DNA and ssf were measured by electron microscopy and compared to that of mature T5 virion DNA. Unexpectedly it was found that both were reproducibly about 12% shorter than the mature molecule; therefore it appears unlikely that the ssf is a true intermediate in the pathway of T5 DNA replication. These observations are discussed in relation to the current models of excision from concatemers and encapsulation of mature phage DNA.

Ultrastructural changes developing in chick embryo fibroblast cultures infected with a wild-type strain of fowl plague virus (FPV) or one of six FPV temperature-sensitive (ts) mutants belonging to different complementation groups were studied. Cells infected with wild-type FPV and incubated at optimal (36 °C) or nonpermissive temperature (42 °C) displayed changes similar to those described for orthomyxoviruses. The same patterns of changes were observed at 36 °C in cells infected with ts mutants belonging to five of the complementation groups. Mutant ts 303, possessing mutation-altered haemagglutinin, induced at 36 °C the formation of virions carrying a considerably reduced number of spikes on their surfaces. At 42 °C, cells infected with ts mutant 131, with a defective primary transcription stage, showed no morphological changes and no formation of electron-dense inclusions. Cells infected with ts mutants with defective secondary transcription or replication displayed nuclear inclusions but no formation of filamentous cytoplasmic structures or virions. Mutant ts 5 with defective late morphogenesis induced formation of considerably enhanced numbers of nuclear inclusions.

The biologically active form of the fusion glycoprotein F from Newcastle disease virus (NDV) comprises two polypeptides, F1 and F2 (derived from a precursor polypeptide F0 by a post translational cleavage event), which are covalently linked together (F1,2) by disulphide bonds. This feature was exploited in a two-dimensional SDS-polyacrylamide gel electrophoretic analysis to orientate the position of the cleavage event within F0. Separation of proteins from NDV-infected CEF in the first dimension in the absence of reducing agent resolved F1,2 protein from all NDV-induced proteins other than F0. Reduction of the first dimension gel with 2-mercaptoethanol, followed by electrophoresis in the second dimension, resolved F1 (55K), F2 (12.5K) and F0 (64K) proteins. The only polypeptides other than F1 and F2 which fell below the diagonal, indicating the positions of the polypeptides from infected cells, were two minor glycoproteins designated HN1 (51.5K) and HN2 (27.5K) which took up positions vertically beneath the major haemagglutinin-neuraminidase glycoprotein HN (75K). Dual isotope labelling experiments with NDV-infected chick embryo fibroblasts, which had previously received a salt shock to effect synchronization of polypeptide initiation upon release of salt shock, revealed the following orientations within the parent molecules: NH2―F2―F1―COOH and NH2―HN1―HN2―COOH.

The existence of intermolecular disulphide bonds, orientation and relative lengths of the two NDV HN fragments is analogous to the HA1 and HA2 proteins of influenza virus haemagglutinin.

The poxvirus Cotia was studied by electron microscopy and by serological and biochemical analyses. Thin-sectioned preparations of infected Vero cells indicated that Cotia virus morphogenesis was similar to other mammalian poxviruses; unique filamentous structures and inclusion matrices were apparent in the cytoplasm. Complement fixation tests that included purified Cotia virions showed a reciprocal cross-reaction with rabbit myxoma virus and no cross-reaction with vaccinia virus. Serological results coupled with gradient polyacrylamide gel electropherograms of the structural proteins of purified Cotia, vaccinia, myxoma and fibroma viruses suggested that Cotia virus was similar to the latter two viruses. Agarose gel electropherograms of cleavage fragments of each of these virus DNAs digested with three separate restriction endonucleases showed that each of these viruses had a unique DNA gel profile.

Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) at concentrations of 10 µg/ml or more, inhibited the replication of infectious pancreatic necrosis virus (IPNV) in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. The drug was most effective when added just before or within 8 h p.i. Incorporation studies with radioactive precursors demonstrated that ribavirin suppressed cellular DNA and RNA synthesis within 2 to 3 h after addition of the drug. The inhibition of nucleic acid synthesis and the antiviral activity was gradually reversed within 3 to 5 days after removal of the drug from the infected cells. Polyacrylamide slab-gel electrophoresis combined with fluorography revealed that: (i) 0.5 µg/ml actinomycin D sufficiently inhibited host cell RNA synthesis thereby enabling the study of virus-specific RNA synthesis in infected cells and (ii) ribavirin inhibited the synthesis of all three virus RNA forms: the transcription intermediate, virus mRNA and progeny dsRNA.

Several B-lymphocyte mitogens have been previously characterized as efficient inducers of endogenous C-type viruses in mouse spleen cell cultures. We now report that foetal calf serum is also capable of inducing C-type virus release in such cultures. While virus induction by B-cell mitogens was found to be serum independent, the combined effects of serum and mitogens were found to be additive and, with some serum batches, synergistic. The kinetics of induction of virus release by serum was very similar to the established pattern using mitogens. The effect of serum was concentration-dependent. The serum lipoprotein fraction prepared by density ultracentrifugation contained virus-inducing activity. By co-cultivation with mink CCL64 cells, stable lines of mouse xenotropic C-type virus could be recovered from cultures which contained serum, serum lipoprotein fraction or mitogens, but not from control cultures. Preliminary evidence indicates that human sera contained a similar virus-inducing activity in the lipoprotein fraction.

We have measured the interferon-inducing particle (i.f.p.) activity of a ts mutant, G11(I), of vesicular stomatitis virus (VSV) and a non-ts revertant, R1 (T1026) in ‘aged’ chick embryo cells and mouse L(Y) cells at 40.5 and 37.5°C, respectively. Our results suggest that a single i.f.p. suffices to induce a quantum yield of interferon and that there are several times more i.f.p. than plaque-forming particles (p.f.p.) in stock preparations of VSV. Furthermore, while virus replication or amplified RNA synthesis is not required for a particle of VSV to induce interferon, there is a requirement for primary transcription. About one-tenth of the genome must remain intact and be transcribed to synthesize an interferon-inducer moiety. (This represents transcription of about two-thirds of the N protein gene.)

We conclude that VSV does not contain a pre-formed inducer of interferon and propose a model for its formation. We suggest that there is a cumulative loss of N (and/or NS and L) protein from the ribonucleoprotein complex during primary transcription, leading ultimately to extensive base-pairing between the genome RNA and its complementary transcript. We suggest that the dsRNA thus formed constitutes the interferon inducer moiety of VSV.

Primary African green monkey kidney cells (GMK) treated with poly(rI).poly(rC) in the presence of DEAE-dextran (‘treated cells’) developed antiviral resistance and concomitantly released interferon into the medium. Treated and untreated cells were infected with herpes simplex virus type 1 (HSV1) in the presence of cytosine arabinoside (araC), and total RNA was isolated and hybridized with purified radio-labelled HSV1 DNA. The intracellular concentration of virus-specific transcripts was not significantly altered in treated cells, but a smaller proportion of the genome of HSV1 hybridized with the extracted RNA. Transcription was similarly restricted when protein synthesis was inhibited by cycloheximide.

To analyse virus translation, proteins were radiolabelled between 6 and 10 h after infection and were immunoprecipitated with a pool of human sera and run on SDS-polyacrylamide gels. No virus-specific proteins could be detected in treated cells. In contrast about 25 HSV1-induced proteins were found in infected cells and about 22 proteins in cells infected in the presence of araC. In particular, two virus proteins with apparent mol. wt. of 128000 and 42500 were immunoprecipitated. Since these two were also detected in cells under conditions where elongation of polypeptide chains was non-specifically retarded, it is unlikely that a similar mechanism was responsible for the impaired growth of HSV1 in our treated cells. We conclude that this impairment probably resulted from regulation at the level of virus translation, probably mediated through interferon.

During ultrahistological investigations of naturally occurring cases of stomatitis papulosa in cattle and Orf in sheep, nuclear changes consisting of aggregations of double membrane-containing tubular structures (outer diam. 100 to 130 nm, inner diam. 50 to 65 nm) and filamentous material were observed. These changes could be reproduced in vitro after infection of bovine (BEL) and ovine (OEL) embryonic lung cell cultures with stomatitis papulosa virus and Orf virus isolates. Nuclear tubules were mostly associated with stomatitis papulosa, whereas filaments were regularly detected in Orf virus infections in vivo. Stomatitis papulosa virus also induced nuclear tubules in vitro in the two cell culture types employed, whereas tubular structures after Orf virus infection only developed in ovine embryonic lung cell cultures in addition to filamentous structures. Orf virus infection of BEL cell cultures induced the formation of filaments. Fluorescent antibody staining revealed parapoxvirus-specific antigens only in the cytoplasm of infected cells.

Cultures of foetal lymphocytes were exposed to u.v.-irradiated herpes simplex virus (HSV). The cells responded with increased 6-3H-thymidine incorporation, the formation of clumps of enlarged lymphoblastoid cells and cell division. This response was first detected 3 to 4 days after exposure to virus material and was shown to be virus-dose dependent. The ability to stimulate foetal cells was considerably more u.v. resistant than infectivity. Two isolates of HSV type 2 (4663 and 37174), which had a high ‘transforming’ ability, produced large numbers of non-infectious particles (particle: infectivity ratios in excess of 104). The cells, which responded to u.v.-irradiated HSV with blastoid transformation, were associated with the non-E-rosetting (T-cell-depleted) subpopulation.

The virus core proteins p30, p15, pp12 and p10 of Rauscher (R-MuLV) and Moloney murine leukaemia virus (Mo-MuLV) were purified. Two-dimensional peptide maps of 3H-leucine-containing tryptic peptides as well as elution profiles from ion-exchange chromatography of tryptic peptides derived from 3H-tyrosine-labelled R-MuLV core proteins and 14C-tyrosine-labelled Mo-MuLV core proteins were compared. The results show that the p30 and p10 proteins are very similar but that p15 and pp12 exhibit significant differences.

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 × 10−6 to 10 × 10−6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.

In street rabies-infected mice, in vivo expression of delayed type hypersensitivity (DTH) measured by the footpad test was revealed by challenge with inactivated fixed rabies virus (RV). The use of BCG as an adjuvant of cell-mediated immunity (CMI) was necessary for the production of significant DTH levels. Typical DTH kinetics were obtained, with a maximum at 24 h after the challenge. DTH was also found to be at highest levels 4 days after infection with street rabies virus. DTH could also be revealed with street rabies virus in RV immunized mice. Adoptive transfer of lymphoid cells from a street rabies infected donor to normal recipient mice was performed and DTH was tested with RV. Susceptibility of DTH to immunosuppression by cyclophosphamide treatment was also assayed in street rabies virus-infected mice and in adoptively-sensitized recipient mice. These results and the relationship between DTH and CMI in rabies infection and immunization are discussed.

An altered baby hamster kidney cell culture which resists the c.p.e. of HVJ (haemagglutinating virus of Japan — the Sendai strain of parainfluenza 1 virus) has been obtained and characterized. These cells, designated BHK-R, were originally obtained by prolonged cultivation of cells surviving HVJ infection; they have been subcultured in the presence of HVJ. No infectious virus was recovered from BHK-R cells and no evidence for the presence of HVJ antigens in the cells was demonstrated by immunofluorescent staining. When BHK-R cells were inoculated with HVJ the growth of challenge virus was suppressed and no obvious cytopathic changes were detected, while these cells normally supported the replication of mumps, influenza, Newcastle disease, vesicular stomatitis or Sindbis viruses. BHK-R cells became susceptible to HVJ infection after serial subculture in growth medium free of HVJ. It was suggested that sialic acid residues present in the surface of BHK-R cell membranes and responsible for adsorption of HVJ were split off by the action of neuraminidase of virus particles, resulting in inhibition of the attachment of challenge virus of HVJ.

Herpes simplex virus (HSV) was isolated from the ears of clinically normal, latently infected mice by culturing the skin in vitro. The results are discussed with reference to current theories of HSV latency.

Proteins associated with 36S virus RNA from Vero cells infected with San Miguel sea lion virus, type 2 (SMSV-2), were labelled with 125I. One protein, VPg, remained linked to RNA when subjected to deproteinization techniques. VPg labelled with 32P was observed on 36S RNA from purified virions; the quantity of label was compatible with two phosphates per genome. The estimated mol. wt. of SMSV-2 VPg was 15000.