- Volume 60, Issue 1, 1982

Ultrathin sections of plaques produced by Streptococcus lactis phages O712 and m13 were examined by transmission electron microscopy. The clear central area of the plaque was found to contain hardly any cellular material but the turbid margin contained abundant plasma membranes and some partially lysed cells whose appearance suggests a novel mechanism for the termination of plaque growth.

Previous studies in this laboratory have quantified the fibrinolytic activity of herpesvirus-transformed cell lines and implicated the proteolytic capacity of cloned cell lines in the formation of primary and metastatic tumours. Because of the involvement of proteases in tumourigenesis in this system, we examined the effect of various protease inhibitors (α-1-antitrypsin, leupeptin and α-2-macroglobulin), as well as hamster serum containing acute phase proteins generated in response to physiological trauma, on the fibrinolytic capacity of a herpes simplex virus type 2-transformed hamster cell line. The effects of the various inhibitors on cell growth, fibrinolysis in vitro and tumourigenesis were examined. Leupeptin, α-1-antitrypsin, α-2-macroglobulin and hamster serum containing acute phase proteins were capable of inhibiting fibrinolysis in vitro, were not toxic to the cells and their action was reversible, while injection of a mixture of protease inhibitor and transformed cells resulted in delayed tumour development.

We have previously shown that adult C57BL/6 mice are relatively resistant to intraperitoneal (i.p.) infection with herpes simplex virus type 1 (HSV-1). Here we show that newborn mice of the strain C57BL/6 are highly susceptible to i.p. infection with HSV-1. Newborn C57BL/6 mice, in contrast to adult mice, did not develop natural killer cell activity in the peritoneal cavity 24 h after i.p. injection of HSV-1, and showed only minimal titres of interferon in the peritoneal fluid after 4 h. After 24 h the peritoneal fluid of newborn mice contained high amounts of interferon and high titres of HSV-1. In contrast, the virus titres in the peritoneal cavity of adult mice were significantly lower. It is suggested that the early titres of interferon at the infection site that are observed in adult, resistant C57BL/6 mice but not in susceptible, newborn mice play a decisive role in resistance.

Treatment of the Burkitt lymphoma-derived cell line Daudi with highly purified human interferon-α (IFN-α) increased up to 100-fold the number of cells expressing Epstein—Barr virus (EBV)-determined early antigen (EA) without inducing the synthesis of virus capsid antigen (VCA), a late virus function. The increase in the number of EA-positive cells was proportional to interferon concentration up to 104 international units (IU)/ml. Treatment of Daudi cells with the same number of units of either partially purified (sp. act. 106 IU/mg protein) or electrophoretically pure (sp. act. 2 × 108 IU/mg protein) IFN-α both gave a similar increase in EA expression, strongly suggesting that the effects observed were indeed due to interferon. Furthermore, treatment of relatively interferon-insensitive Raji cells with IFN-α (1 to 104 IU/ml) had no significant effect on either spontaneous or 5-iodo-2′-deoxyuridine (IdUrd)-induced expression of EA or VCA. Human IFN-β also enhanced the expression of EBV EA in Daudi cells. In contrast, when the latent EBV present in Daudi cells was activated to enter into a replicative cycle, either by induction with IdUrd or by superinfection with the non-transforming P3HR1 strain of EBV, then treatment with human IFN-α caused a marked inhibition of EA expression. Our results suggest that interferon can exert a differential action on virus replication depending upon the state of the virus genome within the cell.

The Burkitt lymphoma/somatic cell hybrid D98/HR-1 contains repressed Epstein—Barr virus (EBV) genomes which can be induced by 5-iodo-2′-deoxyuridine (IdUrd) to express the EBV early antigen (EA) and the virus capsid antigen (VCA). Nuclear and cytoplasmic fractions prepared from uninduced, partially induced and fully induced hybrid cells were radioimmunoprecipitated with human sera having high antibody titres against EA and VCA. Three polypeptides, with mol. wt. 127 × 103, 115 × 103 and 84 × 103 (127K, 115K and 84K), were detected in cells induced to express EA. All three polypeptides continued to be synthesized late in infection. One of the polypeptides was predominantly nuclear, one predominantly cytoplasmic, and one found in both fractions. Three polypeptides, with mol. wt. 144K, 137K and 50K, were detectable only in cells induced to express both EA and VCA. All three polypeptides were found predominantly in the nuclear fraction.

Uncoating of influenza virus (strain WSN) in MDCK cells was studied by following the fate of the virus labelled with radioactive precursors. The accumulation of subviral components of input virus was observed in nuclear-associated cytoplasm (NAC) obtained by treatment of the nuclei with citric acid. Two types of subviral components were found there, ribonucleoproteins (RNPs) and larger subviral particles (SVP) containing RNPs in association with M protein. SVP, with different relative amounts of M protein, were revealed in NAC, suggesting that M protein was gradually released from RNPs. The released RNPs entered the nuclei while M protein accumulated within perinuclear membranes. Thus, SVP could be regarded as probable intermediates in virus uncoating. Rimantadine prevented the release of M protein from RNPs and their penetration into the nuclei provoking the accumulation of subviral components in NAC.

A rimantadine-resistant variant of the Texas strain of influenza virus (Tr) was obtained by serial passages in eggs and in MDCK cells in the presence of the drug, and its uncoating in MDCK cells was compared to that of the sensitive variant (Ts). First and second steps of uncoating were defined respectively by the appearance of subviral particles (SVP) in nuclear-associated cytoplasm (NAC) and ribonucleoproteins (RNPs) in nucleoplasm. In cells infected with Ts, SVP and RNPs were revealed in NAC, while in the presence of rimantadine RNPs were neither found in NAC nor in the nucleoplasm. In cells infected with Tr, SVP but not RNPs were observed in NAC. The amount of RNPs in the nucleoplasm was almost unchanged in rimantadine-treated cells, demonstrating that rimantadine did not interfere with uncoating of the resistant variant. These findings confirm the suggestion that rimantadine blocks the second step of uncoating of sensitive influenza viruses, and are consistent with the idea that this event does account for the prevention of influenza virus infection by the drug.

Purified Sendai virus nucleocapsids isolated from infected cells were used to programme a transcription system in vitro to study virus-specific RNA synthesis. The RNA products were analysed for size by centrifugation before and after denaturation with formamide or glyoxal. The polarity of the products [message (+) or genome (-) strands] was analysed by RNA-RNA hybridization. The non-denatured RNA products sedimented in three groups: 7S to 22S single-stranded RNA transcripts and two partially ribonuclease-resistant complexes. One complex, representing 12% of the total product, sedimented at 26S to 36S. After denaturing the 26S to 36S complex to single-stranded molecules, about half of the RNAs sedimented at 25S to 54S and about half at 6S to 24S. The second complex, representing about 13% of the total RNA product, sedimented at 42S to 52S. After denaturing, about 10% of the single-stranded RNAs sedimented at 38S to 52S and about 90% sedimented at 6S to 19S. In hybridization studies, single-stranded RNAs that sedimented at < 19S were predominantly of message sense (+ strand), whereas RNAs that sedimented at 25S to 54S were a mixture of genome and anti-genome type. These results show that transcription and replication activities in vitro were associated with Sendai virus nucleocapsids obtained from infected cells and that some of the reaction products approached genome size.

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immuno-precipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.

We have isolated and characterized the RNA of intracellular virus nucleocapsids recovered from a number of cell cultures persistently infected with rabies virus or vesicular stomatitis virus (VSV). VSV persistent infections in BHK21, L cells and Aedes albopictus (mosquito) cells generally showed the presence of large amounts of defective-interfering (DI) nucleocapsid RNA and much smaller amounts of standard (B) nucleocapsid RNA. Persistent infections of BHK21 cells by two rabies virus strains, challenge virus standard (CVS-11) or HEP-Flury, were followed for several months during which time the ratio of DI to B nucleocapsid RNA cycled dramatically. We also observed coordinated fluctuations in the absolute amount of incorporation of [3H]uridine into virus nucleocapsid RNA. Total incorporation was generally highest following a decrease in the relative amount of DI nucleocapsid RNA synthesis. At no time were DI nucleocapsids absent in any of the persistently infected cultures.

We have analysed early viral gene products expressed in adenovirus type 12 (Ad12)-infected cells as well as in two Ad12-transformed hamster cell lines, and Ad12-induced rat tumour cell lines by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of virus DNA. Proteins synthesized in vitro were analysed by one- and two-dimensional gel electrophoresis. It was found that RNA encoded by early region E1A directs the synthesis of at least eight polypeptides with apparent mol. wt. 38K, 36K, 30K, 28K, 26K, 25K, 24K and 22K. All these proteins are related to each other. E1B-specific RNA directs the synthesis of three proteins: 59K, 19K and 17K. Early region E2a codes for a 61K polypeptide which probably represents the single-strand DNA-binding protein of Ad12. RNA complementary to region E3 directs the synthesis of a 16K protein, and RNA transcribed from region E4 the synthesis of polypeptides with mol. wt. 20K, 18K and 11.5K. We have mapped a 67K polypeptide into the region within 11 to 28 map units (E2b). The analysis of proteins directed by virus-specific RNAs prepared from two Ad12-transformed hamster cell lines (T637, HA12/7) and one Ad12-induced rat tumour line (RBT12/3) showed that early regions E1 and E4 are expressed in all three Ad12-transformed cell lines. RNA transcribed from early regions E2 and E3 have been detected in lines T637 and RBT12/3. The virus RNA prepared from the Ad12-transformed cell lines directed synthesis of polypeptides with mol. wt. very similar to those of early virus proteins from infected cells. However, in all three Ad12-transformed cell lines mentioned above we have found RNAs which directed the synthesis of additional polypeptides of early regions E1 (34K) and E4 (25K, 24K) not detected in infected cells. The DNA sequence between 11 and 28 map units (coding for the 67K protein) is not expressed in the Ad12-transformed cells.

A virus with morphological and physicochemical properties similar to those of the vertebrate caliciviruses was isolated from navel orangeworms, Amyelois transitella (Walker). Infected larvae contained two types of virus particles: a 185S, 38 nm cupped particle (ACSVi) with a single major polypeptide of 70000 mol. wt. and a 165S, 28 nm smooth particle (ACSVii) with a single major polypeptide of 29000 mol. wt. Larval frass contained a heterogeneous population of virus particles. Evidence is presented which suggests that the 38 nm particle degrades by proteolytic digestion to produce predominantly 28 nm particles in frass. Virus particles contained a single-stranded RNA of 36S (about 2.5 × 106 mol. wt.).

One-hundred and seventeen temperature-sensitive (ts) mutants have been isolated from the challenge virus strain (CVS) of rabies virus (RV). The criterion used for this selection was the absence of plaque-forming units on CER cells under agarose medium at the non-permissive temperature (NPT) of 38.5 °C. Of these mutants, 102 were induced by 5-fluorouracil. This compound was much more effective as a mutagen than either nitrous acid or the alkylating agents ethyl methanesulphonate and methyl methanesulphonate. Seventy-six of the ts mutants exhibited residual multiplication at NPT which was less than 2% of that of the wild-type virus. Attempts to demonstrate complementation were unsuccessful, although three phenotypic groups have been established by biochemical and immunological tests. Possible reasons for the failure to obtain complementation are discussed.

Dense particles (density 1.44 g/ml) were isolated during purification in CsCl of echovirus 11 produced in HeLa cells. The dense particles had similar antigenic properties and similar RNA and protein composition to standard (density 1.33 g/ml) echovirus 11 particles. They differed from standard particles in their higher buoyant density, lower infectivity and slightly smaller diameter. In contrast to other picornavirus dense particles, echovirus 11 dense particles were present as a major component of virus population. This high ratio of dense particles was found only in virus from HeLa cells which produced non-haemagglutinating echovirus 11. Haemagglutinating echovirus 11 from primary monkey kidney (MK) cells did not contain any detectable levels of dense particles.

The tsO45 (V) mutant of vesicular stomatitis virus (VSV) has been used to demonstrate that discrimination between positive (+) and negative (-) strand nucleocapsids in the process of budding mature virions is maintained despite the lack of detectable G protein in either the plasma membrane or the virus membrane. These data indicate that the VSV G protein plays no active role in the discrimination between negative- and positive-strand nucleocapsids which will mature to virions. The synthesis of intracellular nucleocapsids in tsO45 (V)-infected cells at permissive and non-permissive conditions was also examined in comparison with that of the wild-type virus. No differences were observed in intracellular nucleocapsid quantity or polarity in any of these cases despite a 97% inhibition of virus yield in the case of tsO45 (V) at non-permissive temperature.

Human rotaviruses (HRVs) derived from stools were cultivated to high titres in an established cell line (MA-104) using the rotary culture system. Analysis of the 11 double-stranded RNA segments of the culture-adapted HRVs was carried out by polyacrylamide gel electrophoresis. Tissue culture-adapted HRVs, uncloned or cloned, had the same RNA gel patterns as those of the original HRVs from the individual stool specimens. The migrations of the RNA segments from the culture-adapted HRVs were markedly different from that of the RNA segments from calf rotavirus (Lincoln strain). Considerable heterogeneity in electrophoretic migration of the RNA was found among eight strains of the HRVs grown in cell culture. These results confirmed that viruses isolated from stool specimens were indeed of human origin and were not the result of laboratory contamination with tissue culture-adapted calf rotavirus or other viruses.

Using the technique of immune precipitation of [35S]methionine-labelled infected cell polypeptides followed by SDS-polyacrylamide gel electrophoresis and autoradiography it has been shown that SV5 and a closely related isolate are both antigenically related to human parainfluenza virus type 2. Limited cross-reactions were also demonstrated between parainfluenza virus types 1 and 3 by this method and the apparent molecular weights of the major structural components of human parainfluenza virus types 2 and 3 have been deduced.

Avian myeloblastosis virus consists of a mixture of a defective leukaemia virus and several non-defective associated avian leukosis viruses. The genomes of two of the associated avian leukosis viruses were examined in this study and were chosen because one of them, MAV-2(N), induces predominantly nephroblastoma, while the other, MAV-2(O), induces predominantly osteopetrosis. Competitive hybridization studies employing labelled virion RNA and DNA from normal and malignant tissue failed to demonstrate a difference between the genomes. However, examination of ribonuclease T1-resistant oligonucleotide maps revealed that MAV-2(N) RNA had five oligonucleotide fragments which were not present in the MAV-2(O) genome. Poly(A) selection of the oligonucleotides at the 3′ end of the genome showed that the fragments unique to MAV-2(N) were not present at this end of the genome. These results suggest that two viruses differing in oncogenic manifestation also differ in genome composition.