- Volume 66, Issue 4, 1985

In cells productively infected with human cytomegalovirus (HCMV) AD169, large amounts of two viral proteins, the 150K major capsid and the 68K major matrix proteins, are continuously produced during the late phase of infection. In the present study, the mechanism for the intracellular transport of the 150K and 68K proteins was investigated. Infected cells were labelled for 30 min at 72 h post-infection with [35S]methionine, chased for various periods of time at 37 °C, and fractionated into cytoplasmic and nuclear fractions. Immediately after 30 min of labelling, the 68K protein was already associated with the nuclear fraction. In contrast, the major proportion of the 150K protein remained in the cytoplasm for more than 1 h; the migration of the 150K protein was much slower than that of the 68K protein. Both the 150K and the 68K proteins were associated with the perinuclear cytoskeletal fraction in the process of migration. After migration into the nucleus, these proteins were resistant to extraction with DNase and high salt, indicating that they were associated with the nuclear skeleton (nuclear matrix). Effects of various inhibitors on the migration of the 150K protein showed that cycloheximide inhibited the transport of the 150K protein, but other inhibitors such as arabinosyl cytosine, cytochalasin D, colchicine or sodium azide did not. The results suggest that the cytoskeletal structure may play a role in the intracellular transport of HCMV structural proteins from the cytoplasm into the nucleus.

The coding part of a murine interferon alpha (MuIFN-α) gene was cloned into an expression plasmid containing the simian virus 40 early promoter and the rabbit β-globin polyadenylation signal. This construct was transfected into Chinese hamster ovary cells, together with a plasmid containing the Ecogpt gene as a selection marker. Resulting colonies were assayed for constitutive interferon production and analysed for integration of MuIFN-α genes. There was no obvious correlation between the number of genes integrated and the amount of interferon produced. The highest producer, designated CHO-pSV10EF-3, contained four copies of the mouse gene and constitutively secreted up to 100000 International Units of interferon per ml per day. The MuIFN-α subspecies produced by this clone was characterized by analysis of its antiviral activity on heterologous cells, heparin-Sepharose affinity chromatography and chromatofocusing. The results obtained indicate that it is identical or closely related to a minor component present in conventional MuIFN-α preparations.

We have studied the appearance of human interferon-β (HuIFN-β) as well as its mRNA in cells treated with a protein, 22K factor, isolated from the culture supernatant of mitogen-stimulated human peripheral blood leukocytes. By itself 22K was found to be unable to induce production of significant amounts of HuIFN-β protein. However, when aided by treatment with cycloheximide or cycloheximide and actinomycin D (superinduction), 22K caused increases in production ranging from 3- to 20-fold, depending on the cells (diploid or MG-63 osteosarcoma) and the induction schedule. Cells treated with 22K alone produced small amounts of HuIFN-β mRNA, which was only detectable with a highly sensitive method. In combination with cycloheximide, 22K induced levels of mRNA detectable with less sensitive methods as well. These experiments provide further support for the concept that the antiviral activity of 22K is mediated by its ability to stimulate transcription of the HuIFN-β gene in cells.

Adult female Swiss mice showed a greater resistance to intraperitoneal (i.p.) infection with encephalomyocarditis virus (EMCV) than male mice. This difference was not observed in weanling mice, in castrated adult mice or in adult mice injected intracerebrally. Administration of antibody to mouse interferon α/β enhanced the virulence of EMCV for both sexes and no difference was then observed in susceptibility between male and female mice. Six h after EMCV infection, serum interferon titres were higher in adult female mice than in male mice. There was a close correlation between the early serum interferon titre (at 6 h) and survival of EMCV-infected mice. No differences in serum interferon titres were observed between male or female weanling mice or castrated adult mice. Potent preparations of exogenous interferon provided the same degree of protection against EMCV infection in male and female mice. We conclude that the more marked early interferon response of female mice to i.p. EMCV infection is one of the important factors underlying the differential susceptibility to EMCV. It is possible that the interferon system is also involved in the reported greater prevalence of picornavirus infections of men compared with women.

Two interferon-mediated enzymes, a 2-5A synthetase and a kinase that phosphorylates a 67000 mol. wt. (p67K) protein were found at variable levels in different organs of mice. Among the different strains of mice included in this study, germ-free mice had the lowest levels of these enzymes. The levels of 2-5A synthetase and p67K kinase were enhanced significantly in all mice following treatment with mouse (α + β) interferon. Here, we show that the presence of 2-5A synthetase and p67K kinase in different organs of normal mice (untreated) was due, at least in part, to a constant production of interferon under different physiological conditions. Accordingly, injection of normal mice with anti-mouse interferon (α + β) globulin led to a significant decrease in the level of 2-5A synthetase and p67K kinase. In conventional mice (C3H/He), the level of both of these enzymes was higher in female than in male animals and was decreased with age or when such animals were reared isolated in a pathogen-free protected unit. The levels of 2-5A synthetase and p67K kinase were also decreased in normal mice following injection with a powerful antibiotic against a very wide spectrum of Grampositive and Gram-negative bacteria. These results suggest that the production of interferon was induced continuously in normal mice. Such induction was mediated by both internal and external agents.

RNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide. A 5.3 kilobase viral insert in the clone pMB179 has been identified. The region of this clone coding for the spike gene has been sequenced by the chain termination method, and we present here the first report of DNA sequence data for a coronavirus spike protein, the protein which forms the characteristic ‘corona’ after which the group is named. The amino acid sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1162 amino acids with a molecular weight of 127006. This has many interesting features which confirm and extend our knowledge of this recently characterized membrane glycoprotein. The polypeptide is subsequently cleaved to S1 and S2, and partial amino acid analysis of the amino-terminus of the S1 polypeptide has been employed to locate the position of this terminus of S1 within the large open reading frame. The amino acid analysis also reveals the presence of an 18 amino acid putative signal sequence on the primary translation product which is not present on the mature S1 polypeptide.

The so-called M-variant (especially subtype D) of encephalomyocarditis virus (EMCV) induces a diabetes-like syndrome in certain mouse strains which may serve as a model of insulin-dependent diabetes mellitus (IDDM) in man. The development and course of diabetes was influenced by a number of virus and host factors, among these being virus strain, virus dose, mouse strain, age, sex, and the host’s immunological status. In a D-variant stock of EMCV, we found a virus plaque variant (PV 2) diabetogenic for DBA/2 mice, and at least one variant (PV 7) that did not affect carbohydrate metabolism. Although the diabetogenicity of PV 2 proved to be a genetically stable characteristic after further passages in vivo and in vitro, the incidence of diabetes varied somewhat (mean value 65% in 10-week-old DBA/2 mice infected with 105 p.f.u.). Both lower (101 or 103 p.f.u.) and higher (107 or 108 p.f.u.) virus doses led to a diminished incidence and severity of diabetes. In younger animals (5 weeks) transient hyperglycaemia often appeared, whereas in older animals (20 weeks) there was a higher rate of mortality. Histological examination of the islets of Langerhans in diabetes-susceptible (DBA/2) and resistant (C57BL/6) mice revealed that EMCV-induced hyperglycaemia appeared to develop in parallel to islet cell damage. Even in diabetic animals, some unaffected islets were regularly found. This study demonstrates that EMCV mutants may have completely different biological effects and produce diabetes only in special circumstances. Host factors play a significant role in the development of diabetes.

In nearly all systems studied, ribonucleotide reductase consists of two non-identical subunits. We present here the results of our study on herpes simplex virus (HSV) ribonucleotide reductase in favour of the existence of two subunits, H1 and H2, different from the mammalian subunits, M1 and M2. First, although the viral subunits could not be separated by Blue Sepharose chromatography (unlike mammalian subunits), they seemed to dissociate at very low protein concentration as suggested by the non-linear relationship between activity and low protein concentration. Second, pyridoxal phosphate (Pyr.P)-NaBH4 treatment and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) treatment of partially purified extract of mammalian ribonucleotide reductase which inactivated M1 and M2 respectively also inhibited the HSV ribonucleotide reductase. This activity could be restored by mixing Pyr.P-NaBH4-treated extracts with MAIQ-treated extracts of viral ribonucleotide reductase, suggesting that each treated extract contains one active subunit. Moreover, the addition of exogenous M1 and M2 subunits to one or the other of these two treated extracts did not produce any detectable reductase activity. Our interpretation of these results is that the two subunits H1 and H2 which could dissociate upon treatment did not form enzymically active hybrids with the mammalian subunits. Also, the higher degree of resistance to heat inactivation and to hydroxyurea of the viral reductase as compared to the mammalian enzyme suggests that H1 differs from M1 and H2 from M2.

Combined in vivo and in vitro protocols for the generation of anti-murine cytomegalovirus (anti-MCMV) cytotoxic responses were investigated using BALB/c mice and syngeneic mouse embryo fibroblast target cells. Injections of doses of MCMV from 102.6 up to 106.1 p.f.u. into the hind footpad, harvest of draining popliteal lymph node cells after 6 to 8 days, followed by 4 days culture of these cells gave similar and optimal cytotoxic activity against MCMV-infected target cells. After injection of 104.6 p.f.u. into the hind footpad, MCMV was detectable at about 103 to 104 p.f.u. per popliteal node after 3 days, but became undetectable in most animals by 6 days. Cell numbers in the draining popliteal lymph node increased following MCMV inoculation into the hind footpad, but the extent of the increase was inversely related to virus dose and bore no relationship to the anti-MCMV cytotoxic potential of the cells. Lymph node cells applied directly to target cells upon harvest from MCMV-infected mice never gave detectable anti-MCMV cytotoxic activity at 2, 4, 6, 8, 10, 12, 17 or 19 days post-infection, but lysis of uninfected syngeneic targets was obtained 4 to 8 days post-infection.

A cytotoxic response to murine cytomegalovirus (MCMV) was obtained by the culture of lymph node cells from mice inoculated with MCMV into both hind footpads 7 days previously. The cytotoxicity was mediated by Thy1.2+, Lyt2+, H-2-restricted effector cells and was virus-specific. Investigation of the in vitro conditions established that T cell proliferation was necessary for optimal generation of cytotoxicity, that proliferation was dependent upon Thy1.2+, Lyt2+ cell populations and that supernatants from concanavalin A-activated spleen cells enhanced the levels of cytotoxicity obtained.

Permeability changes elicited in Lettre cells by influenza virus at pH 5.3 were maintained when the pH was shifted to 7.4. Permeability changes elicited by Sendai virus at pH 7.4 were maintained when the pH was shifted to 5.3. In each case permeability changes at the new pH were sensitive to inhibition by extracellular Ca2+. The time at which the pH was shifted was critical: if the shift was made prior to the onset of permeability changes, no subsequent changes occurred. It is concluded that the pH-sensitive event, namely virus-cell fusion, is related to the induction of permeability changes through attainment of some type of ‘threshold’ level of membrane damage.

By inoculating chickens intranasally with a collection of strains of infectious bronchitis virus (IBV) of the Massachusetts serotype and of Escherichia coli of different serotypes, a pool of viral and bacterial strains was selected which, on inoculation, consistently produced a highly lethal disease closely resembling the natural disease produced by these two organisms. The conditions for reproducing the experimental disease were not rigorous in that, within broad limits, the size of the viral and bacterial inocula were not important; neither were the times at which both organisms were administered in relation to each other. The breed or strain of chicken used was important and the resistance of chickens to fatal infection increased with age. When the E. coli strains of the pool were inoculated intranasally without the IBV component, the chickens remained well; bacteriological examination of chickens inoculated with one of the E. coli strains, O18, revealed little evidence of invasion of the tissues or even of persistence of the inoculated E. coli strain in the upper respiratory tract. A minority of the IBV strains examined were lethal for chickens when inoculated without E. coli but many of them only produced a substantial mortality when the E. coli were included in the inoculum; IBV strains in this latter category included the vaccine strains H52 and H120. High concentrations of IBV strain M41 and E. coli O18 persisted in the upper respiratory tract for a number of days after they had been inoculated together. Much lower concentrations of IBV M41 were found in the internal organs, such as the spleen; E. coli O18 was only found in these sites in some of the inoculated chickens. Coliform organisms proliferated in the upper respiratory tract of chickens inoculated with IBV alone; they were rarely found in their internal organs.

The pattern of both constitutive and interferon-induced proteins was determined by two-dimensional gel electrophoresis in parental and interferon-resistant clones of Daudi cells in relation to the phenotype of interferon resistance. The complement of constitutive proteins present in clones DIF3, DIF8, DIF9 and DIF10 appeared to be identical to that of parental Daudi cells even though these cells were resistant to both the antiviral and anti-proliferative actions of interferon. Treatment of Daudi cells for 20 h with 103 reference units/ml of electrophoretically pure human interferon-α resulted in the induction of 15 proteins of molecular weights ranging from 15000 to 62000 detected after a 4 h labelling period with l-[35S]methionine. A number of these proteins were also induced in interferon-resistant clones of Daudi cells although some of these proteins appeared later and in smaller amounts than in the interferon-treated parental cells. However, seven proteins with molecular weights ranging from 18000 to 58000 which were induced by interferon in parental Daudi cells were not induced in any of the four interferon-resistant clones, suggesting that the phenotype of interferon resistance of these cells may be related to a reduction or absence of certain interferon-induced protein(s).

The genetic and antigenic variation in 12 Sindbis (SIN) virus isolates from four zoogeographic regions (Paleoarctic, Ethiopian, Oriental and Australian) has been examined at a molecular level. RNase T1 oligonucleotide fingerprinting of genomic RNA from SIN isolates revealed that the primary structure of the RNA from viruses from each zoogeographic region was unique. The E1 and E2 glycoproteins and the capsid protein of two isolates from each zoogeographic region were compared by tryptic peptide mapping with the Egyptian prototype strain AR-339. Tryptic peptide maps of viruses from Sicily and the Ethiopian region were similar to those of the prototype; maps of isolates from the Oriental and Australia regions were different from each other and from those of the prototype strain. Viruses from each of the four zoogeographic regions were analysed antigenically by neutralization with polyclonal serum to AR-339 and by enzyme-linked immunosorbent assay with an anti-E2 monoclonal AR-339 antibody. Clear antigenic divergence of SIN isolates into two groups, representing the Paleoarctic-Ethiopian and Oriental-Australian regions were demonstrated. These results support a hypothesis which proposes that ancestral SIN virus diverged into two distinct groups. The genetic changes have resulted in further phenotypic divergence within the geographic varieties.

Monoclonal antibodies were developed that are specific for Rous sarcoma virus structural, polymerase (reverse transcriptase) and transforming proteins. The monoclonal antibodies were shown to bind to purified virus proteins in an indirect 125I-labelled Protein A binding assay suitable for screening even very large numbers of hybridomas. Additional tests for specificity included radioimmunoprecipitation of purified virus structural proteins P12 and P27, of reverse transcriptase subunits α and β, and of the transforming protein pp60v-src. Pilot immunofluorescence and protein kinase assays of the expression of virus proteins in avian and mammalian cells infected by wild-type virus as well as by temperature-sensitive, transformation-defective virus mutants revealed that synthesis of virus structural and transforming proteins is hardly affected by changes in temperature, whereas the pp60v-src-associated kinase activity is temperature-sensitive in cells infected by most, but not all the virus mutants.

Density gradient fractions prepared from healthy or scrapie-infected hamster brain tissue enriched in plasma membrane vesicles were treated with nucleases prior to phenol extraction and ethanol precipitation. The recovered nucleic acids were 3′ end-labelled and run on one-dimensional polyacrylamide gels. Autoradiography revealed the presence of low molecular weight RNAs (4S) in both healthy and scrapie samples. Two-dimensional fingerprint analysis indicated that the RNAs isolated from scrapie-infected hamsters contained oligonucleotides that were not present in RNAs isolated from healthy hamsters.

The sensitivity of the scrapie agent to u.v. inactivation was found to be related to the purity of the tissue preparation. Scrapie infectivity associated with membrane vesicles was unaffected when irradiated with 104 J/m2. Irradiation of more highly purified preparations from detergent-extracted CsCl gradient fractions reduced scrapie infectivity from 107.8 log10 LD50 per ml to as low as 104.5. Sensitivity of membrane-associated scrapie infectivity to inactivation by u.v. irradiation could be increased by addition of chlorpromazine, a phenthiazine antipsychotic which penetrates lipid bilayers and induces single-strand breaks in nucleic acids under irradiation. Chlorpromazine without irradiation, and a semiquinone protein-binding radical of chlorpromazine, failed to decrease scrapie infectivity by themseleves. A closely related phenthiazine antipsychotic, trifluoperazine, which does not bind to nucleic acids, did not reduce scrapie infectivity. These findings suggest that the target of u.v. radiation for inactivation of scrapie infectivity in the presence of chlorpromazine is an essential nucleic acid.

Previous studies have shown that the scrapie agent is highly membrane-associated. We examined the protein composition of gradient fractions enriched for large membrane vesicles prepared from scrapie-infected and uninfected hamster brain using various methods to extract membrane proteins. We also examined proteins in detergent-extracted membrane vesicles fractionated on CsCl gradients. No qualitative differences in protein composition were seen comparing scrapie-infected and uninfected samples by one-dimensional gel electrophoresis. Extraction of proteins from membrane vesicles by phenol, pyridine, perchloric acid or lithium diiodosalicy-late also failed to reveal any unique proteins in scrapie-infected hamster brain. Attempts to solubilize hydrophobic proteins (proteolipids) from CsCl gradient fractions into organic solvents were unsuccessful. These findings indicate that any hydrophobic protein associated with the scrapie agent is not a proteolipid, and that the ability of solvents to reduce scrapie infectivity is not a result of extraction of a proteolipid.