- Volume 60, Issue 2, 1982

We have previously described several series of biochemical transformation experiments in which small defined portions of herpes simplex virus (HSV-1 and HSV-2) DNA encompassing the thymidine kinase (TK) gene were introduced into Ltk− cells by the calcium transfection procedure. The presence of authentic virus TK enzyme in several subcloned cell lines derived from these experiments was confirmed by either the specific incorporation of [125I]iododeoxycytidine into their nuclei or the inhibition of cell growth by the antiviral drug arabinosyl thymine. A panel of 24 independent Ltk+ cell lines receiving either isolated virus DNA fragments or cleaved plasmid DNAs was examined by blot hybridization for both the presence and copy number of virus TK DNA sequences. Most cell lines contained a single virus DNA fragment covalently joined to host (or carrier Ltk−) mouse DNA sequences, but several contained multiple copies of the TK gene. Examination of the structural arrangement of the virus DNA in two early passage multicopy cell lines indicated that the TK gene had integrated into Ltk− cell DNA and then subsequently both viral and flanking cellular sequences were amplified to create up to 20 tandem duplications. In one case, mapping of the adjacent cellular sequences has revealed that the total repeat unit is greater that 23 kilobases (kb) in size. On subsequent passaging, even in HAT medium, the amplified repeat units were not stable and fell to only three to four copies per haploid cell genome. These cell lines should prove useful for additional studies to examine the expression of co-selected non-TK virus sequences and the influence of adjacent cellular DNA sequences on transcription and retransfection of the resident TK gene.

Athymic nude (nu/nu) mice were inoculated in the ear pinna with 104 p.f.u. herpes simplex virus type 1 (strain SC 16). Initially, the virus was observed to replicate in the pinna, spreading via a neurological route to the dorsal root ganglia, spinal cord, brain and adrenal glands. Following the transfer of lymphoid cells from day 7 herpesvirus-infected hairy immunocompetent donors into infected nude mice, virus was not isolated from the pinna and nervous system of the majority of the mice. The passive transfer of neutralizing polyclonal anti-herpesvirus serum or neutralizing monoclonal anti-gp D serum did not reduce infectivity in the pinna, but markedly reduced the amount of virus in the ganglia and spinal cord. These data suggest that neutralizing antibodies play an important role in restricting the movement of virus to the nervous system, whereas cell-mediated immune (CMI) mechanisms are essential for eliminating virus from the pinna.

Using electron microscopic autoradiographic techniques, the uptake of [methyl-3H]thymidine into uninfected and guinea-pig cytomegalovirus (GPCMV)-infected guinea-pig embryo (GPE) fibroblast cells was investigated. In GPCMV-infected GPE cells, [methyl-3H]thymidine uptake into cellular DNA was inhibited prior to the onset of virus DNA synthesis as well as during the entire period of virus replication. Virus DNA synthesis increased slowly during the period of active virus replication, which usually lasted from 18 to 48 h post-infection. Only the electron-dense amorphous matrices and fibrillar structures in the nuclear inclusions were associated with the tritiated labels and thus contained virus DNA.

Tracheal organ cultures infected with mouse cytomegalovirus (MCMV) were shown to produce virus for up to 180 days with virus replication primarily in epithelial cells. Persistent virus infections were established in tracheal organ cultures from both MCMV-susceptible and MCMV-resistant strains of mice. In acutely infected tracheal organ cultures, cellular DNA synthesis appeared to precede the production of significant amounts of virus antigens and the release of virus into culture fluids. Since persistently infected tracheal organ cultures continued to synthesize cellular DNA, the results suggest that host cell turnover may continually renew the population of MCMV-susceptible cells. The results suggest a possible mechanism for virus persistence in tracheal organ culture based on continuing serial infection of newly susceptible cells combined with prolonged release of virus from infected cells.

In human fibroblast cells treated with interferon, cytomegalovirus-specified immediate early RNA was found associated with the polyribosomes at concentrations and size classes similar to the virus RNA found in non-treated cells. Interferon treatment inhibited the translation of the immediate early virus mRNA; the relative rate of virus-specified immediate early protein and antigen synthesis decreased with increasing concentrations of interferon. In addition, the relative amount of virus-specified RNA associated with the polyribosomes at early times after infection was significantly reduced by treatment of the cells with interferon. Inhibition of infectious virus production in interferon-treated cells was primarily due to inhibition of immediate early virus protein synthesis and secondarily to suppression of early virus RNA synthesis. The role of the virus-specified immediate early proteins in regulating subsequent virus gene expression is discussed.

To facilitate a molecular analysis of Shope papilloma virus-induced neoplastic cells, we have established a cell line from Vx-7, a transplantable tumour originally induced by the Shope virus. Single phase molecular hybridization and Southern transfer methods were employed to assess copy number and physical state of the DNA, and the extent of transcription. Both tumour and cell line were found to contain multiple copies of the virus genome and these were all integrated into the host cell DNA. Transcripts corresponding to a complexity of approx. 1% of the virus genome were detected at low abundance. These results are discussed relative to our earlier findings with tumours induced directly by virus, and to requirements for maintenance of the Vx-7 tumour over the 30 years that it has been in existence.

The human adenovirus serotype 12 (Ad-12) EcoRI-C DNA fragment (0 to 16.5 map units) was cloned in the plasmid vector pAT153. This cloned Ad-12 EcoRI-C DNA fragment was subcloned generating recombinant plasmids which contained the Ad-12 SalI-C fragment (0 to 10.3 map units), the Ad-12 HindIII-G fragment (0 to 6.8 map units) and the Ad-12 AccI-H fragment (0 to 4.7 map units). Thus, we constructed recombinant plasmids which contain Ad-12 DNA sequences which represent all or part of the virus transforming gene region. The capacity of the cloned Ad-12 EcoRI-C DNA fragment to transform rat cells in vitro was assessed using the focus assay on primary cultures of rat cells. The specific transforming activity of this recombinant plasmid was in the same range as that found for intact Ad-12 DNA. Transformed foci which were induced by the cloned Ad-12 EcoRI-C DNA fragment were established as cell lines and the presence of Ad-12 DNA in these lines was demonstrated using the Southern blotting technique.

High resolution polyacrylamide gel electrophoresis (PAGE) of chick embryo fibroblast cells infected with the avian influenza virus FPV-Rostock revealed two distinct polypeptides migrating in the region of the nucleoprotein (NP). One-dimensional fingerprinting of these polypeptides showed that they were both nucleoprotein, and [32P]orthophosphate labelling revealed that they differed with respect to their state of phosphorylation. Pulse-chase studies using [35S]methionine indicated that phosphorylation of a certain proportion of NP occurs rapidly after synthesis and is associated with transport to the nucleus. Nucleoprotein which remained in the cytoplasm was predominantly non-phosphorylated. Both the phosphorylated and the non-phosphorylated types of NP were found in ribonucleoprotein complexes (RNPs) of different densities isolated on renografin gradients, but RNPs isolated from the nucleus contained much more phosphorylated NP than those from the cytoplasm. The kinase responsible for nucleoprotein phosphorylation appears to be influenced by temperature of incubation of the infected cells.

Evidence for the replication of human coronavirus strain 229E (HCV 229E) in macrophages is presented. Virus antigen was detected in macrophages by an immunofluorescent technique 24 h after infection and virus particles were observed in the cisternae of the endoplasmic reticulum by electron microscopy. Giant cells were observed by light and scanning electron microscopy, and large multinucleate cells were seen by thin-section electron microscopy, suggesting that HCV 229E can induce syncytial formation in cultured human macrophages. Furthermore, the production of infectious virus by macrophages was demonstrated by an infectious centre assay.

The pattern of protein synthesis in HeLa cells simultaneously infected with encephalomyocarditis virus (EMCV) and Semliki Forest virus (SFV) has been analysed throughout the time course of the infection. The ratio of the picornavirus protein γ versus the togavirus late protein C increased when the m.o.i. of EMCV was raised, and the ratio of C/γ increased with higher multiplicities of SFV. Under some conditions, the co-infected cells simultaneously synthesized the picornavirus and togavirus proteins, and the cells exclusively translated the capped 26S mRNA from SFV at the end of the co-infection experiment. The influence of the time of addition of the second virus on the relative translation of the capped and uncapped mRNAs was also studied. When HeLa cells were co-infected with 5 p.f.u./cell of EMCV and 200 p.f.u./cell of SFV, only the synthesis of SFV proteins was apparent, whereas if SFV was added 1 to 3 h later during the course of EMCV infection the cells synthesized picornavirus and togavirus proteins. If the cells were superinfected with SFV 1 h after EMCV addition, host protein synthesis was drastically inhibited after 3 h of EMCV infection. By 5 h post-infection both kinds of virus proteins were synthesized and at 7 h post-infection the cells preferentially translated the capped 26S mRNA from SFV. If cells were first infected with SFV (10 p.f.u./cell) and co-infected or superinfected at 1 h with EMCV (50 p.f.u./cell), the shut-off of host protein synthesis occurred 3 h after infection and the cells synthesized both kinds of virus proteins. However, 9 h after infection the cells synthesized SFV proteins only. When double-infected HeLa cells were placed in a hypotonic medium, they mainly synthesized the togavirus late proteins, whereas under hypertonic conditions, they translated the picornavirus RNA exclusively. These results suggest that the two kinds of mRNAs (SFV 26S mRNA and EMCV 35S mRNA) are present in the infected cells and that the relative translation of each of them depends on the external ionic conditions.

The way in which ultraviolet-irradiated vesicular stomatitis virus (VSV) inhibits the early events in VSV infection has been further characterized. Comparison of several different u.v.-irradiated thermolabile, temperature-sensitive mutants before and after heat inactivation established a requirement for inhibitory activity of functional G, N and L proteins, but not M protein. Defective-interfering (DI) particles, whether irradiated or not, inhibited VSV primary transcription as efficiently as UV-VSV, suggesting that virus proteins rather than transcription products are responsible for inhibition. Addition of inhibitory UV-VSV at different times after infection established that inhibition results from an action at an intracellular site, rather than at the cell surface or in the process of internalization. A similar inhibition by UV-VSV of infection by Sendai virus, Semliki Forest virus, Sindbis virus and influenza virus suggests that UV-VSV is acting by inducing a general change in the intracellular environment.

Formalin-fixed Staphylococcus aureus strain Cowan, bearing protein A, routinely used for the absorption of antigen-antibody complexes, was found to bind protein kinase activity from disrupted Moloney murine leukaemia virus (Mo-MuLV). The Wood strain of S. aureus lacking protein A also bound the kinase with similar efficiency. About 50% of the bound kinase activity, as detected by phosphorylation of casein using [γ-32P]ATP, could be eluted from the bacterial preparation with buffer containing 0.5 m-KCl. Similar results were obtained with Moloney murine sarcoma virus (Mo-MuSV) strain 349 and ts110 MuSV(MuLV). The bacterial preparation was also found to bind casein kinase activity from cellular extracts of uninfected, Rauscher murine leukaemia virus (R-MuLV)-infected and Mo-MuLV-infected cells. Analysis of [3H]leucine-labelled proteins from purified virus showed selective binding to S. aureus of only two major labelled virus proteins. One virus component bound to S. aureus had the relative mobility of p15; the other polypeptide co-migrated with virus p10. Upon exposure to increased salt concentration, most of the p10 but very little of the p15 proteins were released. The S. aureus-binding proteins from ts110 Mo-MuSV and MuSV-349 revealed similar binding and elution patterns of p10 and p15 molecules. The p10 and protein kinase activity eluted from Mo-MuLV-absorbed bacteria were separated by gel filtration into a high molecular weight species, containing p10 and kinase activity, and a low molecular weight p10 monomer lacking enzymic activity.

DNA was extracted from varicella-zoster (VZ) virions prepared in sucrose gradients. Thirty-eight molecules examined by electron microscopy were found to have a mean length of 46.7 µm. Examination of self-annealed VZV DNA molecules revealed that the virus genome was composed of a unique linear large sequence with a mol. wt. of 74.4 × 106 to 78.4 × 106, and a unique short sequence of mol. wt. approx. 9.8 × 106 flanked by inverted repeat sequences of 4.7 × 106 mol. wt.

A Marek’s disease lymphoblastoid cell line (MSB-1) has been analysed by immunoprecipitation for expression of tumour-associated antigen, Marek’s disease virus (MDV)-specific antigens and antigens specific to avian leukosis-sarcoma viruses. Rabbit antisera raised against two independently derived cell lines after extensive absorption with normal chick cells reacted with a polypeptide of mol. wt. 40000 (40K) in extracts of MSB-1 cells. The 40K polypeptide was not present in myeloblasts or in chick embryo fibroblasts (CEF) infected with MDV and did not react with antiserum raised against normal chicken thymus antigens. The possibility that the 40K polypeptide is a tumour-associated antigen is discussed. Seven MDV-specific antigens were noted in infected CEF (mol. wt. 110K, 100K, 80K, 70K, 50K, 35K and 32K) but none of these was detected in MSB-1 cells. The avian leukosis-sarcoma group-specific antigen P27gag and its precursor Pr76gag were not found in MSB-1 cells, confirming that expression of mature gag protein is not required for transformation by MDV. However, two polypeptides of unknown origin and function (mol. wt. 180K and 110K) were precipitated from MSB-1 cells with a rabbit anti-Rous sarcoma (Schmidt-Rupin, subgroup D) antiserum.

A non-structural protein of mol. wt. 34 × 103 was demonstrated in the nuclei of Rift Valley fever virus-infected Vero cells by SDS-polyacrylamide gel electrophoresis. The protein appears to correspond to the virus-induced antigen demonstrated by indirect immunofluorescence in intranuclear inclusions.

Human embryonic lung fibroblast cultures and Vero cell cultures infected with cell culture-adapted strains of human coronavirus (HCV) OC43 or neonatal calf diarrhoea coronavirus (NCDCV) were shown to possess highly cross-reactive membrane-associated antigens (MAA) by the indirect fluorescent antibody technique (IFAMA). MAA appeared 3 h post-infection, concurrently with the appearance of cytoplasmic antigens. Electron microscopic observations of cell cultures infected with either coronavirus strain and labelled with the immunoperoxidase antibody (IPA) technique for MAA detection showed that MAA consisted mainly of a strongly labelled, discontinuous, brush-like layer of amorphous material, strictly associated with the infected cell membrane. By light microscopy, reactivity of MAA with homologous and heterologous immune serum was similar to that of antigens detected by IPA in ethanol-fixed infected cells. IPA and IFAMA, but not haemagglutination-inhibiting (HI) and neutralizing (Nt) antibody, were strongly decreased by absorption of immune sera with trypsin-treated glutaraldehyde-fixed cell cultures infected with homologous virus. MAA IgG antibodies were detected by IFAMA in both human and animal sera. Sera from infants showing an HI and Nt, but not an IPA, antibody response to HCV OC43 were also free of detectable IFAMA antibody to HCV OC43.

Peripheral blood lymphocytes of chickens bearing tumours induced by avian sarcoma virus can be specifically stimulated to divide by the crude culture fluids of virus-infected cells. In this communication, we show that relevant antigenic activity apparently resides in each of the internal virus proteins p15 and p27. The ability of infectious culture fluids to be mitogenic for sensitized lymphocytes is greatly reduced following treatment with antibodies specific for either total avian myeloblastosis virus (AMV) protein or for p27.

The susceptibility of human leukocyte (α), fibroblast (β) and recombinant α-2-interferons to clearance by the isolated and perfused rabbit liver has been evaluated. Human leukocyte and recombinant α-2-interferons were stable and their initial levels were maintained in the perfusate even if they had been treated with neuraminidase, thus suggesting that α-interferons have no exposed sugars recognizable by hepatic binding proteins. On the other hand, native, and particularly desialylated human β-interferon, underwent marked hepatic uptake confirming the importance of the liver as a catabolic site for glycosylated interferons.

Vero cells persistently infected with parainfluenza virus type 3 (para 3) were examined for the production of temperature-sensitive (ts) mutants. After 6 months and 33 passages, ts mutants formed the great majority of the virus being shed into the supernatant fluid. Complementation studies gave evidence that the mutants shared a common lesion. Although ts mutants may play a role in the maintenance of persistent infection in this system, the mutants proved to be unstable when removed from the milieu of persistent infection. It is thus possible that their selection may be secondary to (an) unidentified factor(s) which play(s) a more primary role in the maintenance process.