- Volume 64, Issue 10, 1983

The genome profiles of 200 field isolates of bluetongue virus were obtained by extracting the double-stranded RNA genome from each isolate and separating the ten genome segments by polyacrylamide gel electrophoresis. These virus isolates, consisting of representatives of the four bluetongue virus serotypes existing in the U.S.A., were obtained during an epidemiological study commencing in 1979 in four western states of the United States. Analysis of the migration patterns of the ten double-stranded RNA genome segments revealed distinct variations of electrophoretic profiles of many isolates. These variations are discussed in relation to the year and geographical region in which the isolate was made, the species of animal from which the virus was obtained and the electropherotype relationships of the four bluetongue virus serotypes existing in the U.S.A.

Two strains of human parainfluenza virus 2 (HPV2), P2 1972/6 and P2 1980, grow to high titre in MEK3 cells, and their structural proteins and virus-induced protein synthesis have been characterized by gel electrophoresis and immunoprecipitation. Purified viruses contain seven polypeptides, including cellular actin: L (175K mol. wt.), HN (72K to 74K), NP (66K to 67K), F1 (52K to 58K), P (49K), A (44.5K) and M (39K). Virus-induced polypeptide synthesis was first detected at 8 h post-infection with the appearance of NP; other major structural proteins were detected from 10 to 12 h after infection and onwards. The synthesis of both the structural glycoproteins was demonstrated, although proteolytic processing could not be detected. Reproducible differences in the gel migration of the HN, F1 and NP polypeptides were found in whole virus, in infected cells and cells subjected to immunoprecipitation. These differences may reflect genetic diversity within HPV2 and provide a means of probing the molecular epidemiology of these viruses.

The arginine carboxypeptidase involved in the proteolytic cleavage of the haemagglutinin of influenza A virus has been analysed by an assay employing a Sepharose-bound peptide containing radioactive arginine as a substrate. The enzyme activity has been extracted from purified virus with non-ionic detergents and has been separated from the haemagglutinin and from the neuraminidase by isoelectric focusing and by affinity chromatography. The carboxypeptidase present in virus grown in different host cells shows variations in its isoelectric point. It can be concluded from these observations that the carboxypeptidase is a host component incorporated into the virus envelope. When the enzyme is inhibited by 2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid, haemagglutinin with the arginine attached to the carboxy terminus of HA1 can be obtained. The observation that under these conditions the haemagglutinin has retained its haemolytic activity indicates that the carboxypeptidase does not play an essential role in the activation process.

Mixed infection of MDCK cells with influenza A and influenza B viruses leads to a reduction in the rate of synthesis of haemagglutinin (HA) and nucleoprotein (NP) as compared to their rate of synthesis in cells separately infected with these viruses. The reduction is much stronger for influenza A virus proteins. The synthesis of the nonstructural NS1 protein of both viruses is relatively resistant to the heterotypic interference. The synthesis of virus-specific mRNAs exhibits the same pattern: the formation of the transcripts of HA and NP genes is much more drastically reduced than the synthesis of NS gene transcripts. The effect is strongly dependent on the multiplicity of infection and on the ratio of influenza A and B viruses in the inoculum. Primary transcription in the presence of cycloheximide is almost unchanged in doubly infected cells as compared to single infection, and no indication of differential inhibition has been observed. The results are discussed in connection with the mechanism of heterotypic interference and the regulation of influenza virus protein synthesis.

The envelope of the bunyavirus La Crosse contains two glycoproteins, G1 (120000 mol. wt.) and G2 (38000 mol. wt.). When incubated with trypsin or plasmin, the G1 glycoprotein of virus grown in cell culture was cleaved, leaving two different sized polypeptides in the envelope (67000 and 95000 mol. wt.). Chymotrypsin cleaved G1 leaving polypeptides of 70000 and 100000 mol. wt. G2, however, was not altered by these enzymes. When used in antibody neutralization studies, these proteolytically modified viruses were neutralized approximately 1 to 2 log10 units in 60 min while control virus was neutralized by over 4 log10 units in 20 min. Because antibody to G1, but not G2, was involved in La Crosse virus neutralization, cleavage of G1 appeared to be directly responsible for these altered kinetics of neutralization. Antibody did bind to the polypeptides remaining associated with the envelope resulting in infectious virus-antibody complexes. This indicated that a critical site in terms of antibody neutralization was removed from G1 by proteolytic enzymes.

Previous work by M. J. Buchmeier and his colleagues and by our group has led to the conclusion that the lymphocytic choriomeningitis (LCM) virus contains seven distinct structural proteins, which we have named p200, gp85, p77, p63, gp60, gp44, and gp35. Their arrangement in the virion has now been analysed by establishing nearest-neighbour relationships with a homobifunctional crosslinker, by performing polyacrylamide gel electrophoresis in parallel under reducing and non-reducing conditions, and by determining the proteins that are covalently bound to viral lipids. A hypothetical model of the virion of LCM virus is proposed. Its envelope is assumed to consist of a membranous layer composed of gp60 and lipids and two types of spikes with either gp85 or gp44 as tips and gp35 as bases. The last-mentioned glycoprotein also appears to be complexed with p63, the main protein component of the nucleocapsid, and this in turn was found to be spatially associated with p200. Probably p77 is also an internal component, but a more exact position cannot yet be assigned to this protein.

Five rat strains were studied for immune responsiveness to SFFV-NRK, a normal rat kidney cell line non-productively infected with spleen focus-forming virus (SFFV) of the Friend leukaemia virus (FLV) complex. Antisera from ACI, JAR, Fischer and SD strains precipitated SFFV-specific gene product, gp55, whereas those from LEW did not. In the cross of Fischer × LEW, immune responsiveness to gp55 was controlled by a single or a few dominant genes. The immune responsiveness was neither related to the susceptibility to FLV infection, nor to the amount of SFFV-related RNA in spleen cells. Unresponsiveness of LEW rats to gp55 was not absolute; when LEW rats were immunized with FLV preparations from infected mice or xenotropic virus-infected NRK cells, they produced antibody that could precipitate gp55.

A highly leukaemogenic virus isolate (DMBA-LV) endogenous to the CFW/D mouse has been found to contain two viral genomes. One was closely related to the type B milk-borne mouse mammary tumour virus (MMTV) and present in tenfold excess over a type C viral genome which was only partially related to xenotropic and polytropic isolates from the CFW/D mouse as well as to the ecotropic Moloney murine leukaemia virus isolate. The thymic lymphoma cell line that produced DMBA-LV expressed high levels of MMTV viral RNA (35S and the 24S envelope mRNA). Both the virus and the virus-producing cell line expressed multiple species of type C viral RNA. Similar species of type C viral RNA were also associated with non-infectious, non-leukaemogenic viral particles present in both normal lymphoid cells and in a MMTV-free thymic lymphoma cell line established from a second chemical carcinogen-induced tumour.

The stabilities of B77 avian sarcoma virus intracellular RNAs were compared to the stability of the total cellular poly(A)-containing RNA by labelling infected chicken embryo fibroblasts with [3H]uridine for 15 h, adding actinomycin D (1 µg per ml) to block further transcription of viral RNA, and selecting virus-specific RNA from the total cellular poly(A)-containing RNA at 3 hourly intervals. The three virus-specific RNA species (9.3, 3.3 and 5.4 kilobases) decayed with half-lives of 7.5, 10, and 15 h, respectively, whereas the bulk of the cellular mRNA decayed with a half-life of 13 h. To correlate these decay rates with the disappearance of mRNA activities, the actinomycin D-treated cells were pulse-labelled with [3H]leucine at 3 hourly intervals after the addition of the drug and virus-specific protein synthesis was assayed by immunoprecipitation. The mRNA activity for the precursor to the non-glycosylated viral structural proteins (Pr76gag) decayed with a half-life of approximately 6 h, whereas the mRNA activity coding for the precursor to the envelope proteins (gPr92env) decayed with a half-life of 14 h. Thus, the rate of decay of the individual mRNA species corresponded reasonably well with the decay rate for the synthesis of two of the corresponding gene products. The results indicated that the 5.4 kb env mRNA is more stable under these conditions than the 9.3 kb gag mRNA but was not significantly more stable than the bulk of the cellular mRNA. Virus particle production following the addition of actinomycin D was determined by the reverse transcriptase assay and by the incorporation of viral genomic 70S RNA into extracellular virions. Both assays yielded similar results and indicated that particle production was inhibited at a rate (t(math) = 4 h) somewhat faster than the decay of Pr76gag synthesis or the disappearance of 9.3 kb RNA. It was established by two independent methods (pulse and chase, and approach to isotope equilibrium), however, that the intracellular half-life of the RNA that is packaged into virions is 6 to 7 h. Thus, these results suggest that a single metabolic pool of 9.3 kb RNA exists in avian sarcoma virus-infected cells and is used both as mRNA and as genome RNA.

The stability of P85gag-mos, encoded by ts110 Moloney murine sarcoma virus, was studied. P85gag-mos was found to turn over more rapidly at 39 °C than 33 °C in two different cell lines. One of these is a non-producer cell clone (6m2) which appears only to express P85 at 33 °C. The other cell line (206-2IC) produces P85gag-mos at both 33 °C and 39 °C, although in lower amounts at the higher temperature. Both cell lines exhibit the normal morphological phenotype following a shift to the restrictive temperature. These data suggest that the presence of P85gag-mos alone is insufficient to cause transformation, and that some function(s) associated with the molecule may be heat-labile.

RD-114 is a human sarcoma-derived cell line which is chronically infected with the RD-114 retrovirus. In a previous study, we found that treatment of these cells with human interferon-α or human interferon-γ causes a marked inhibition of RD-114 virus production, but that the replication of exogenous vesicular stomatitis or encephalomyocarditis virus is not impaired. In the present study, we report that neither type of interferon has strong inhibitory effects on DNA synthesis or on multiplication of the cells. We also failed to detect a double-stranded RNA-dependent protein kinase activity in extracts of both interferon-treated and untreated cells. However, a low level of 2′,5′-oligoadenylate [2,5(A)] synthetase activity was detectable in extracts of interferon-treated cells. 2,5(A)-dependent endonuclease L activity was detectable in extracts of both untreated and interferon-treated cells. This was probably responsible for the inhibition of protein synthesis observed upon introduction of 2,5(A) to RD-114 cells. In many cells, interferon has been found to induce synthesis of several proteins demonstrable by autoradiographic analysis of slab gels on which extracts of interferon-treated and radiolabelled cells are separated. Using a similar method, no such induced protein synthesis was detectable in interferon-treated RD-114 cells. Our results indicate that RD-114 cells are resistant to most known actions of interferons except for the antiretroviral action to which they are as sensitive as any other cell line.

A simple solid-phase immunoassay for quantification of vesicular stomatitis virus (VSV) is described. Infected cultures are lysed with deoxycholate. Samples of the lysates are transferred to PVC immunoassay plates and the amount of virus protein adsorbed to the plates is then quantified by sequential incubation with antiserum against VSV proteins and 125I-labelled Protein A. The decrease of VSV protein in interferon (IFN)-treated cultures is correlated with inhibition of formation of infectious virions; its quantification therefore allows accurate measurement of the antiviral effect. The applicability of the immunoassay for measuring the virus yield is not restricted to cells exhibiting a virus cytopathic effect. Moreover, since the decrease of virus protein is obtained at IFN concentrations lower than those that reduce cell killing by the virus, the assay provides a more sensitive measure for the IFN effect than that obtained by ‘cytopathic effect inhibition’ assays.

DNA polymerase activity present in Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus-infected Spodoptera frugiperda cells has been analysed by chromatography on DNA-cellulose and phosphocellulose columns. In infected cells a new fraction of activity was found to bind to the columns more strongly that did polymerase activity in uninfected cells. The infected-cell-specific DNA polymerase was purified by a combination of DNA-cellulose chromatography of crude extracts and phospho-cellulose chromatography of the semi-purified activity. The final product contained a single polypeptide of molecular weight 126 000 which was not found in uninfected cells. The purified enzyme was inhibited by aphidicolin and [E]-5-(2-bromovinyl)-2′-deoxyuridine triphosphate, but not by bromovinyldeoxyuridine. The enzyme was shown to be an early enzyme, probably a delayed early protein, since it was present in cells inhibited by aphidicolin which were locked into the synthesis of early proteins by the drug.

Two herpesvirus isolates from goats are known which cause afflictions of the digestive tract in kids and, in some cases, abortion. An antigenic relationship of these goat herpesviruses with infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus (bovid herpesvirus 1, BHV-1) was reported and because of the species-specific pathogenicity, the goat isolates were named caprine herpesvirus 1. In this report the two isolates are further characterized and compared with BHV-1. Although the caprine herpesviruses share many biological and physicochemical properties with BHV-1, they can be differentiated from the bovine viruses with respect to growth cycle, one-way cross-neutralization and, most importantly, the restriction endonuclease fragments of their DNAs. The molecular weight of the caprine herpesvirus DNA, based on electron microscopic length measurement is 90 × 106, similar to that of BHV-1 (95 × 106). On the basis of these genomic differences, we propose that DNA restriction endonuclease patterns of the caprine herpesviruses should be designated as prototypic of bovid herpesvirus 6 (BHV-6).

The alkaline nucleases induced by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been purified from high salt extracts of virus-infected cells. The purification used three types of column chromatography and resulted in apparently homogeneous DNase preparations with good recovery. The enzyme from HSV-2-infected cells has been characterized. It had both exonuclease and endonuclease activity, each with an unusually high pH optimum. The enzyme had an absolute requirement for magnesium which could not be replaced by other divalent cations. Analysis of the sedimentation characteristics and electrophoretic properties of the purified enzyme indicated that it was composed of a single subunit of mol. wt. 85000. The purified HSV-2 enzyme was used as an immunogen to prime BALB/c mice which were used to prepare monoclonal antibodies. Three monoclonal antibodies were shown by several criteria to react with the enzyme. Thus, we were able to confirm that the 85K polypeptide did indeed have nuclease activity. This polypeptide was designated ICSP 22 in earlier studies and is a major polypeptide of virus-infected cells.

Sequences from the whole of the HSV-1 strain 17 genome were cloned into bacterial plasmid vectors, with the exception of part of BamHI v which was deleted in all cloned DNAs spanning this region of the virus DNA. The cloned DNAs were used in intratypic marker rescue experiments to map temperature-sensitive (ts) mutations on to the virus genome. Since the sequences of these DNAs overlapped, any mutation could be rapidly assigned a physical map position. This approach is particularly useful for mapping spontaneous mutations and lesions induced by mutagenesis of whole virus DNA. In this study, we mapped ten ts mutations comprising eight different complementation groups. Five lesions, representing three different cistrons, were located within BglII k (map units 0.098 to 0.166), and three mapped within EcoRIf (map units 0.321 to 0.414), two of which were in previously unidentified cistrons of HSV-1 strain 17. One mutation analysed had a defect within the short repeat region and another had a mutation within EcoRI i (map units 0.632 to 0.720).

After several serial passages at low multiplicities of infection in primary human foetal glial cells at 37 °C, the DNA of prototype (MAD-1) JC virus and that of MAD-2 and MAD-3 are typically heterogeneous in size, but DNAs of MAD-4 and MAD-6 are relatively homogeneous. A similar dichotomy was observed in the DNAs of six isolates propagated more recently in glial cultures at 39 °C under similar conditions of brief passage in vitro at low multiplicities of infection: the DNAs of two (MAD-9 and -10) were heterogeneous, but the DNAs of four others (MAD-8, -11, -12 and -14) were homogeneous. Therefore, the propensity of the viral genome to sustain deletions was an intrinsic property of each isolate. However, actual induction and maintenance of the presumably defective DNAs was influenced by the relative proportions of permissive spongioblasts and semi-permissive astrocytes in the glial cultures and by the multiplicity of infection. Deletions in MAD-1 DNA were confined to the presumptive early region and spanned the BamHI cleavage site (map position 0.505). The heterogeneity was more complex in the DNAs of MAD-2 and MAD-3, but again most of the deletions, which ranged up to 12% of full-length DNA, spanned the BamHI site. We propose that the differential susceptibility to deletion among isolates is a consequence of natural genetic variation in JC virus.

Hepatitis B virus (HBV) DNA was found to be integrated into seven sites in the DNA of the PLC/PRF/5 hepatoma cell line as determined by digestion with the restriction endonuclease HindIII which does not cut through the viral genome. The integration pattern was stable in the cell line, in tumours induced in athymic mice by this line and in cell lines derived from such tumours. Syntheses of hepatitis B surface antigen and alphafoetoprotein were maintained in the induced tumours and derived cell lines. A defective HBV DNA molecule (approx. 2.8 kilobase pairs) appears to be integrated in a head-to-tail tandem arrangement and it is proposed that such defective molecules may be involved in the process of neoplastic transformation by HBV.