- Volume 58, Issue 2, 1982

To study the effect of gene dosage on gene expression, λplac5cI857O29P3, a replication defective lambda phage carrying part of the lac operon (containing the lac promoter, operator and z gene) in the b2 region was studied in Escherichia coli strain JC6256 where the lac operon is deleted and at a temperature where the λ repressor is inactive. In measuring the synthesis of β-galactosidase, it was possible to separate the effects of the lac promoter from those of the phage promoter. When the synthesis of β-galactosidase was initiated from the inserted lac promoter in JC6256(λ+) in the presence of additional cyclic AMP, the rate and level of β-galactosidase synthesis were directly proportional to the multiplicity of infection (gene dosage). Furthermore, β-galactosidase synthesis was initiated about 5 min after infection, just as with isopropyl-β-d-thiogalactoside (IPTG) induction.

When the synthesis of β-galactosidase was initiated from the phage promoter in JC6256 in the absence of additional cyclic AMP, the rate and level of β-galactosidase synthesis were again linearly proportional to gene dosage. On the other hand, initiation of β-galactosidase synthesis was delayed until 10 to 20 min after infection. These results suggest that: (i) in the absence of negative controlling factors, the extent of gene expression is proportional to gene dosage; (ii) varying the gene dosage can be used to regulate gene expression.

To measure the length of heating required to convert a λ lysogen in the immune (im+) phase into the anti-immune (im−) phase, rex gene activity was used as an indicator. It was observed that 5 min heating at 41 °C did not shift any lysogenic cells of 594(λN − cI857O −) from the im+ phase into the im− phase, and it took 17 min heating at 41 °C followed by long hours of culture at 30 °C to shift half of the lysogenic cells into the im− phase. Such a length of heating is too long to be accounted for by blocking of the expression of lambda repressor by cro gene product. The result is more consistent with accumulation of a certain level of cro gene product during heating so that the synthesis of the repressor is blocked even after a return to low temperature.

The replication of unselected strains of herpesvirus saimiri (HVS) was sensitive to bromodeoxyuridine and bromovinyldeoxyuridine (BVdU) but insensitive to acycloguanosine (ACG), in contrast to the growth of herpes simplex virus (HSV) which was sensitive to all three analogues. Mutants of HVS resistant to bromodeoxyuridine and BVdU could be selected by growth in the presence of these inhibitors. Productive infections of owl monkey kidney or Vero cell cultures by unselected strains of HVS resulted in increases in a thymidine kinase (TK) activity which was deficient in cells infected with bromodeoxyuridine-resistant mutants of the virus. Induction of the virus enzyme promoted a net increase in the uptake and incorporation of exogenous labelled thymidine in the face of the progressive inhibition of the overall incorporation of [35S]methionine and [3H]uridine into productively infected cells. The TK induced in cells infected with HVS differed from the major activity of uninfected cells and resembled that encoded by HSV in its capacity to phosphorylate iododeoxyuridine and in the sensitivity of all the thymidine phosphorylating activity to competition by BVdU. However, in contrast to the HSV TK, which phosphorylated deoxycytidine and iododeoxycytidine relatively efficiently and was sensitive to ACG, the HVS enzyme did not phosphorylate deoxycytidine or iododeoxycytidine and was insensitive to ACG. Whilst HVS, therefore, shares the characteristic of other members of the herpesvirus group of inducing a novel TK, the properties of the HVS-induced enzyme differ significantly from the enzyme of the prototype herpesvirus, HSV. The properties of the HVS TK are nonetheless sufficiently distinct from those of the uninfected cell to provide a possible basis for selective antiviral chemotherapy based on preferential phosphorylation of nucleoside analogues such as BVdU by infected cells.

Cross-neutralization tests were done on accepted species and recently isolated members of the genus Orthopoxvirus using antisera which had been separately absorbed with the various viruses. The results provided evidence for the involvement of four neutralizing antigens, and their distribution among 13 virus strains was determined. Monkeypox (Congo-8-Lombe), camelpox (Gorgan), ectromelia (Mill Hill), ‘Lenny’ and elephant poxviruses had distinctive antigenic formulae. Lister and Wyeth vaccines were indistinguishable but different from Copenhagen and EM63 vaccines which were themselves distinct. Cowpox (Brighton), buffalopox (BP4), MK10, and Moscow poxviruses were indistinguishable. Examples were found where viruses shared surface antigens but were not all neutralized by antibody to them. This reduced the practical value of the technique for virus identification. Evidence was also obtained for the existence in some viruses of a fifth antigen, antibody to which could block neutralization by antibody to one particular antrgen.

Infectivity of nucleic acid from highly infectious mouse scrapie brains was studied by transfecting the nucleic acid in vitro prior to inoculation into animals. Foetal mouse brain and whole mouse embryo cultures were chosen as the cells used for the transfection. As an internal control, infectious nucleic acid was recovered from bacteriophage φX174 and whole virus particles were obtained when cultures were transfected with herpes simplex virus DNA. In contrast, none of the animals inoculated with nucleic acid preparations derived from scrapie-infected tissues developed scrapie disease either with or without transfection procedures. These findings (i) show transfection techniques fail to elicit evidence of a scrapie-specific infectious nucleic acid and (ii) confirm the observation that scrapie is not a viroid.

The antibody and HLA-restricted virus-specific cytotoxic T lymphocyte (CTL) responses to influenza vaccine of 36 volunteers were analysed. Three vaccines were used: a live attenuated, and two types of inactivated, a whole virus and a purified surface antigen vaccine. Antibody to haemagglutinin (HA) was assayed using plaque neutralization, single-radial-haemolysis and haemagglutination-inhibition (HI) techniques. Antibodies to nucleoprotein and matrix antigens were also measured. Most of the volunteers had antibody responses to the HA in the inactivated vaccines which were detected by all three techniques. Nine of the twelve recipients of the live virus vaccine did not have an antibody response detected by the HI test, but four of these did have antibody responses when the plaque neutralization test was used. Single-radial-haemolysis was more sensitive than the HI test for detecting low levels of antibody, but the plaque neutralization test was the most sensitive for detecting low levels of antibody. Most volunteers had a rise in their HLA-restricted influenza-specific memory CTL response, but three recipients of live vaccine who did not have an antibody response by any technique also did not have an increase in their cytotoxic T cell activity. Three volunteers, two of whom had received live vaccine, had a positive CTL response without antibody response.

Monoclonal antibodies having three different specificities have been prepared against the Egypt 101 strain of West Nile virus (WNV). All were reactive against the envelope glycoprotein in radioimmune precipitation tests, and all enhanced WNV replication in the mouse macrophage line P388D1. One antibody, which is of IgG2a subclass, had strong neutralizing activity against the homologous virus, although it failed to neutralize a different WNV strain. The other two antibodies, both of IgG1 subclass, had little neutralizing activity, but one had strong reactivity with the haemagglutinin of the inducing virus and other flavivirus haemagglutinins.

The antigenic specificity of antibody-dependent plaque enhancement (ADPE) on the macrophage cell line P388D1 has been investigated using viruses in the flavivirus and alphavirus genera. Whereas ADPE detected group-specific antigenic determinants in the flaviviruses, narrower specificity (type and subgroup) was seen with alphaviruses. ADPE titres correlated well with haemagglutination inhibition titres.

The effects of microtubule-depolymerizing agents on virion production in MJD-54 cells chronically infected with Moloney murine leukaemia virus were examined. By measuring the amount of reverse transcriptase activity remaining in particles recovered from culture fluids, we found that incubation with 1 µm- or 10 µm-colchicine, vinblastine or nocodazole resulted in 30 to 40% decreases in virus production. The decrease in virus production did not seem to be due to general damage to the cells since cellular RNA and protein synthesis were only slightly, if at all, inhibited by the drug treatment (<10%). Furthermore, virus proteins accumulated inside drug-treated cells, viz, [35S]methionine-labelled Pr65gag showed a 1.5-fold increase over a 3 h continuous label interval. Consistent with this accumulation of virus protein, electron microscope studies showed that inside drug-treated cells there was a 2- to 2.5-fold accumulation of virions within cytoplasmic vesicles. All of these results support the idea that cytoplasmic microtubules play a role in the production of murine leukaemia virus.

Chick syncytial virus (CSV), a strain of avian reticuloendotheliosis virus (REV) causes lymphoid tumours in chickens after a prolonged incubation period. A number of CSV-induced tumours were examined for cell surface antigen and were found to be of the B cell type and to produce immunoglobulin. Attempts were made to grow in vitro cell lines from CSV-induced tumours and a lymphoblastoid cell line was established from a liver tumour of a chicken that was inoculated with CSV via the yolk sac in embryo. The donor chicken was viraemic at the time the tumour was removed. The cell line is designated RECC-RP13, it produces non-defective REV, is a B cell type and it produces IgM. It is free from infection with endogenous and exogenous avian leukosis virus (ALV) and has an increased number of chromosomes. Sequences specific to REV were detected in at least four sites in cellular DNA from RECC-RP13. Sequences specific to ALV DNA, beyond that normally found in 15I5 × 71 cells, were not found in DNA from this cell line.

The characteristics of chinook salmon embryo cells persistently infected with infectious pancreatic necrosis virus were consistent with defective-interfering (DI) particle-mediated persistence. All the cells were infected and were slowly releasing virus, but they could be cured of virus in the presence of antiserum. Immunofluorescence showed that the amount of virus antigen in persistently infected cells was low. This fact, coupled with the observation that few DI particles were released by these cells, indicated that DI particles were not replicated to excess in this cell line.

Influenza viruses, which had lost up to 99.999% infectivity by incubation with antibody (α) specific for the haemagglutinin (HA) or with monoclonal α-HA, attached on to and penetrated chick embryo fibroblast (CEF) cells to the same extent as non-neutralized virus. Neutralized virus was also uncoated efficiently as shown by the accumulation of virion RNA in the nucleus and virion envelope in the cytoplasm. Polyacrylamide gel electrophoresis of virion RNA segments recovered from the nucleus or cytoplasm of cells inoculated with neutralized or non-neutralized virus showed that antibody did not potentiate degradation of RNA. However, these RNAs were not expressed since virus-induced proteins were not detected in cells to which neutralized virus had been added. Assay of virion transcriptase of neutralized virus in vitro showed that its activity was reduced up to sevenfold compared with non-neutralized virus, and annealing studies showed that no detectable transcription took place in vivo with neutralized virus. These studies support the conclusion that antibody directed specifically against the HA protein on the outer surface of the influenza virus particle neutralizes infectivity by inactivating virion transcriptase activity and it is suggested that antibody to HA brings about allosteric rearrangements in the HA molecule which are transmitted across the virus envelope to the interior of the particle.

Facets of virulence for ferrets of 16 recombinant clones of two parent viruses A/Finland/4/74 (H3N2) and A/Okuda/57 (H2N2) were determined and viewed in relation to their genetic composition. Of the five pairs of recombinant clones with RNA segments of the same parental origin, differences in facets of virulence were detected between members of two of the pairs. One pair differed in ability to produce fever, and another pair in ability to infect the lower respiratory tract. Subsequent analyses indicated slight differences in two genes of the first pair of viruses. Of all the recombinant clones examined the three that derived their surface antigens from the attenuated parent (A/Okuda) were less virulent than the 13 which possessed the surface antigens of the virulent parent (A/Finland). The virulence of the latter clones tended to decrease as the number of RNA segments that were derived from A/Okuda increased.

BHK cells infected with strain 17 syn + (HSV-1) or HG52 (HSC-2) incorporated inorganic sulphate into polypeptides which co-migrated on SDS-polyacrylamide gels with virus-induced glycoproteins. The major sulphated glycoprotein was glycoprotein E. In addition, less-intense sulphated bands co-migrated with glycoprotein D and HSV-1 glycoprotein A/B/C. Sulphate label co-migrating with HSV-2 glycoprotein A/B/C was occasionally observed. We have investigated which sulphated polypeptides are excreted from infected cells. Major ones of apparent mol. wt. 32000, 34000 and 35000 were excreted from cells infected with 17 syn +. In addition, polypeptides which migrated in the vicinity of glycoprotein D were often excreted from cells infected with either 17 syn + or HG52. The 32K, 34K and 35K polypeptides were antigenically related to glycoprotein D and over 95% of the total amount synthesized was excreted. Analysis of intracellular sulphated polypeptides using intertypic recombinants mapped glycoprotein E to between 0.832 and 0.950 units of the HSV genome.

The surface alterations of HEp-2 cells induced by mixed infection with two different picornaviruses (poliovirus and ME virus) were compared by scanning electron microscopic and transmission electron microscopic studies and by 51Cr-release assay. The contribution of each of the viruses to the resulting surface changes was discernible, as investigations on the chronology of the cytopathic alterations demonstrated that the changes were distinct for either virus. The surface of ME virus-infected cells was characterized by large membranous structures (‘sheets’ and blebs) representing huge vacuoles. These sheets were not seen in poliovirus-infected cells. Poliovirus induced more prominent cell pycnosis, elongation of filopodia and condensation of collapsed microvilli on the cell surface than ME virus. Mixed infection with these two viruses led to surface alterations typical for ME virus. These ME virus-specific changes occurred irrespective of poliovirus reproduction or its inhibition by guanidine. ME virus-specific alterations also predominated in cytolytic membrane damage as expressed by 51Cr-release from infected cells. 51Cr-release was more pronounced from ME virus than from poliovirus-infected cells, even when ME virus reproduction was suppressed by interfering poliovirus. However, alteration of the internal structures of the infected cells was only dominated by ME virus when the reproduction of poliovirus was suppressed.

The kinetics of synthesis and the metabolic stability of uncapped vesicular stomatitis virus (VSV) mRNA transcripts have been studied using techniques which clearly differentiate them from other RNA species. The triphosphate-initiating mRNA transcripts accumulate for at least 5 h during a typical in vitro transcription reaction. The great majority of these transcripts are smaller than a functional message and have been released from the template–transcriptase complex. Label that accumulates in them is stable and is not detectably diminished after a 1 h chase with unlabelled precursor. These kinetic properties are not those expected for active intermediates in mRNA synthesis and suggest that the uncapped transcripts are products of aborted transcription that accumulate during the transcriptive process. However, we cannot rule out that a small subset of these transcripts may be elongated into mature mRNA. Initiation of transcription at internal positions along the VSV genome is both frequent (one-half to one-sixth as frequent as initiations at the leader RNA gene) and site-specific (occurring only at the beginning of cistrons). The relevance of these results to the models for VSV transcription is discussed.

We have constructed a hybrid plasmid molecule that contains the complete coding sequences from the 26S mRNA of Semliki Forest virus. Five fragments which together covered the mRNA sequence were isolated from three original hybrid plasmids and joined together at five different restriction endonuclease cleavage sites using T4 DNA ligase.

Antibodies were raised in rabbits against the structural components of human coronavirus strain 229E and mouse hepatitis virus strain 3, prepared from disrupted virus particles. Hyperimmune sera to the subcomponents showed cross-reactions by enzyme-linked immunosorbent assay between ribonucleoprotein antigens of these viruses, indicating the presence of a common antigen(s). None of the other virus structural components showed any cross-reactivity.