- Volume 62, Issue 1, 1982

The structural proteins of Rhizobium meliloti temperate phage 16–3 have been analysed by means of polyacrylamide gel electrophoresis, isoelectric focusing and agarose gel electrophoresis. Five major and five minor proteins were identified and characterized with respect to their size, isoelectric point and their distribution between the head and tail of the phage particle. The synthesis of structural proteins was studied by one- and two-dimensional gel electrophoresis.

The nucleotide sequence of 4 kilobases of DNA from within the short region of the genome of herpes simplex virus type 1 has been determined. This portion of DNA contains the junctions of the terminal and inteRNAl inverted repeat sequence components with the unique sequence component and the 5' regions of the genes which encode the Vmw 12, Vmw 68 and Vmw 175 immediate-early polypeptides. The transcription and translation initiation sites of all three genes and the 5' and 3' boundaries of the Vmw 12 and Vmw 68 gene introns have been localized on the DNA sequence and shown to be flanked by sequences which resemble those found in similar positions in other eukaryotic genes. For the Vmw 12 and Vmw 68 genes the promoters, the 5'-non-coding regions of the mRNAs, and the introns lie within the terminal and inteRNAl inverted repeats respectively while the polypeptide-coding regions lie in the short unique component. The introns consist largely of tandemly reiterated copies of a 22-nucleotide sequence: the Vmw 12 gene intron contains seven of these and the Vmw 68 gene intron five. The Vmw 175 gene is located entirely within the short repeats, of which those regions sequenced here have the extremely high G + C content of 78%, in marked contrast to the value of 66% obtained for the adjacent region of the unique sequence component. Prediction of the complete amino acid sequence of the Vmw 12 polypeptide, accounting for a mol. wt. of 9830, and of the first 523 amino-terminal amino acids of the Vmw 175 polypeptide has been made from the DNA sequence. The polypeptide-coding region of the Vmw 175 gene has an average G + C content of 80% but nevertheless specifies a wide range of amino acid types because of the maximal assignment of G and C residues to codon third base positions.

Herpes simplex virus type 1 (HSV-1) induces an immediate-early (IE) polypeptide IE 12. An equivalent polypeptide coded by HSV-2 which migrated slightly more slowly on SDS-polyacrylamide gels was identified and designated IE 12.3. Analysis of the serotype of the IE polypeptide induced by five HSV-1/HSV-2 intertypic recombinants when correlated with their genome structures showed that IE 12/IE 12.3 mapped in the region spanning the TRS/US junction. Unlike four other IE polypeptides induced by HSV-1 (IEs 175, 110, 68 and 63), IE 12 was not detectably phosphorylated.

Several triphosphates (TP) of 5-substituted deoxyuridine (dU), like 5-ethyl (Et), 5-n-propyl (n-Pr), 5-iso-propyl (iso-Pr), 5-n-hexyl (n-Hx), and 5-trifluorothymidine (F3-dT) were used as substrates for HeLa DNA polymerase α and for two herpes simplex virus (HSV)-coded DNA polymerases isolated from HeLa cells infected with HSV-1, strain C42 (wild-type), or its mutant resistant to phosphonoformate (PFA r). All polymerases were purified up to the DNA-cellulose column step and they showed comparable specific activities. The incorporation into DNA studied with all the alkyl analogues of dUTP is several times higher with the virus enzymes than with DNA polymerase α. The DNA polymerase of the mutant virus incorporates dUTP analogues to a lower extent than the wild-type polymerase. The two virus enzymes also differ in the K m and V max values for different substrates, indicating that the mutation to PFAr has affected the structure of the virus DNA polymerase. Surprisingly, all three enzymes use F3-dTTP as substrate for DNA synthesis to an equal but limited extent.

The in vitro growth of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts (MEF) from Balb/c, C57BL, C3H and CBA mouse strains has been shown to correlate with the genetically determined in vivo susceptibility of these strains to MCMV infection. MEF from Balb/c and C57BL mice were more susceptible to MCMV than those from C3H and CBA mice. This was evident regardless of whether replication was measured by cytopathic effect (c.p.e.) score, virus yield, plaque count, plaque size or time of onset of c.p.e. The growth of MCMV in tracheal organ cultures from different mouse strains was similar to that observed in MEF from these strains. The replication in MEF when measured by c.p.e. score and virus yield was affected by the density of the cell cultures. The strain of mouse used to produce MCMV also affected the comparative sensitivity of MEF to the virus. This appeared to be due to reduced growth of MCMV in its homologous MEF type, an unexpected result. In contrast, cell density had little effect on the replication of herpes simplex virus type 1 (HSV-1), but again the in vitro susceptibility of MEF reflected the in vivo susceptibility of mouse strains to infection with this virus.

The intracellular precursor polyproteins of simian sarcoma-simian-associated virus [SiSV(SiAV)] were compared to the intracellular proteins of the human retrovirus isolates, HL23V, HEL12V and A1476V, by radioimmunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and tryptic peptide analysis. Cells infected with SiSV(SiAV) were characterized by polyproteins Pr200gag-pol, gPr80env, Pr80gag, Pr60gag and Pr40gag. Identical intracellular precursor polyprotein profiles were obtained from cells infected with HL23V, HEL12V and A1476V. Tryptic digest mapping of peptides containing [3H]leucine showed the structural composition of Pr60gag to be the virus core proteins, p28, p15/p12 and p10. The SiSV(SiAV) envelope precursor, gPr80env, contained the structural determinants of mature viral gp70 and a non-glycosylated protein termed p15E. The homology of the human isolate viruses, HL23V, HEL12V and A1476V, to the SiSV(SiAV)/GaLV (gibbon ape leukaemia virus) family of viruses was confirmed by mapping studies. Both gPr80env and Pr60gag of SiAV were identical by tryptic peptide mapping to the respective proteins from the three human retrovirus isolates examined. The potential significance of these results to considerations of the origins of SiAV and the SiAV-like human isolates is discussed.

The effects of interferon treatment on the expression of murine mammary tumour virus (MuMTV) by a continuous mammary tumour cell line of GR mouse were investigated. Interferon markedly inhibited the excretion of MuMTV particles in the culture media as measured by hybridization of virus-specific RNA and virus-associated reverse transcriptase activity. There was no inhibition of virus RNA and protein synthesis in those conditions. The steady-state level of intracellular virus RNA and its rate of synthesis were not modified by interferon treatment. The intracellular levels of the major virus envelope and core proteins as measured by immunoprecipitation techniques remained unchanged. Interferon failed to inhibit the synthesis and the processing of the gp52 glycoprotein and p27 core protein precursors. However, the rate of maturation of the glycoprotein precursor was slowed down. Surface labelling of intact cells did not reveal accumulation of virus proteins on the cell membrane upon interferon treatment. The intracellular level of MuMTV-characteristic reverse transcriptase activity was reduced in interferon-treated cells, although to a lower extent than extracellular virion-associated enzyme activity. MuMTV particles released from interferon-treated cells revealed no difference in their protein composition. These results are consistent with an inhibition by interferon of a late step in the replication of MuMTV.

Discrete subgenomic DNA fragments were found in three out of thirty-two preparations of adenovirus type 2 incomplete particles grown in human Hep-2 cells and examined over the course of 1 year. One preparation contained three fragments corresponding to 5, 14 and 19% of the genome, another contained a 37% fragment and the third a 40% fragment. Each fragment hybridized exclusively to the left end of the genome. Digestion of the nick-translated 37% fragment with HindIII confirmed that it contained the left 37.3% of the genome. Synthesis of these fragments was not dependent on high input multiplicity of infection. Comparable fragments were not found in unpackaged DNA from the corresponding infected cells. This is consistent with the hypothesis that such fragments are generated during virus assembly or, alternatively, may reflect the very small proportion of these fragments relative to the pool of unpackaged DNA within the cells. The possibility that they are generated by errors in DNA replication is discussed.

Addition of 50 μg/ml chloroquine to neuroblastoma cells 1 h before infection with temperature-sensitive mutant ts G31 (III) of vesicular stomatitis virus (VSV) prevented virus-induced cell fusion from occurring. Interestingly, addition of chloroquine after infection still inhibited cell fusion. Based on the number of fusion events required to produce the polykaryocytes observed, cell fusion was inhibited 92% when chloroquine was added 1 h post-infection and 77% when chloroquine was added 2 h post-infection. The inhibition of virus-induced cell fusion could not be accounted for by an inhibition of virus protein synthesis because the virus protein synthesis measured 6 h post-infection was 90% of that in untreated, infected cells with chloroquine added 1 h post-infection, and the same as untreated, infected cells when chloroquine was added 2 h post-infection. No virus proteins were made, however, when chloroquine was added before infection, which is consistent with a chloroquine-mediated inhibition of virus uncoating. The release of infectious virions was completely inhibited when chloroquine was added before infection or 1 or 2 h post-infection, which indicated an inhibition of virus maturation in the later stages of virus assembly. By indirect immunofluorescence the virus glycoprotein (G protein) could not be detected on the surface of chloroquine-treated, infected cells, but the G protein was present inside the treated cells. With 125I-labelled anti-G protein IgG, 16% of the G protein found on the surface of untreated, infected cells was on the cell surface when chloroquine was added 2 h post-infection. When chloroquine was removed from infected cells, the G protein accumulated at the cell surface, and this accumulation could not be prevented by tunicamycin, an inhibitor of glycosylation. Furthermore, galactose was incorporated into the G protein in the presence of chloroquine. Therefore, the VSV G protein was being synthesized and glycosylated in the presence of chloroquine but the drug prevented the expression of the glycoprotein at the cell surface during the final stages of G protein assembly.

The inner capsid layer of human rotavirus was found to preferentially break up into large ring-like morphological units. The outer capsid was found to be composed of capsomeres covered by a thin protein or glycoprotein covering. These capsomeres appeared to be broad headed and short stemmed, similar to the type of pin used to mark locations on a map (pushpin).

A canine virus derived from a diseased dog has been plaque-purified and characterized in detail. Analysis of infected cells demonstrated that virus antigen accumulated in the nucleus at 12 to 24 h post-infection and the cytopathology at the ultrastructural level was diagnostic of a parvovirus infection. The purified virus particles were 23 to 26 nm in diam. and banded at a density 1.44 g/ml in CsCl. Detailed biochemical analysis revealed a single-stranded DNA genome and three structural proteins of mol. wt. 82300, 67300 and 63500. All of the data presented are consistent with the classification of this virus as a parvovirus.

A flame photometric technique is described for determining average values for intracellular [Na+] and [K+] in HeLa cells. Ion measurements were made on unwashed cells disrupted ultrasonically in the presence of residual medium; corrections for the latter were made by measurement of extracellular volume in cell plus medium preparations using 125I-labelled polyvinylpyrrolidone (PVP) as the marker in an isotopic dilution technique. Accurate measurement of the volume occupied by the cells was critical and required a concentration step. This was achieved by concentrating cell suspensions in a microhaematocrit centrifuge using calibrated capillary tubes. Most reliable values were obtained in our system using HeLa S3 (suspension) cells grown as monolayers, which were removed by EDTA and held in suspension for a minimum of 2 h. Uninfected HeLa cells had values of 20 to 30 and 110 to 120 mM for Na+ and K+ respectively. At 13 h after inoculation with vaccinia virus, a dramatic reversal in [Na+] and [K+] occurred, but throughout the infection cycle the total [Na+ + K+] varied little. The significance of these data is discussed in relation to theories of virus-induced modulation of protein synthesis in infected cells and in cell-free systems.

To investigate the DNA sequence homology between the multicapsid nuclear polyhedrosis virus of Autographa californica and the multicapsid and single capsid nuclear polyhedrosis virus of Orgyia pseudotsugata, the stringency of hybridization conditions was varied. Thirteen to 25 % homology between the multicapsid viruses of A. californica and O. pseudotsugata was detected in 20, 30 and 40% formamide, indicating that fairly stable duplexes were being formed. Under the most stringent conditions in 50% formamide little homology was detected. In contrast to the multicapsid viruses, the single capsid virus of O. pseudotsugata demonstrated about 10% sequence homology in 20% formamide, but these duplexes were unstable and the percentage homology decreased markedly in the higher formamide concentrations. Examination of the specific regions of homology by hybridizing labelled DNA to Southern blots of the virus DNAs revealed that the regions of homology between these viruses were not limited to one region of the genome but were found on a number of restriction fragments.

A panel of 125 monoclonal antibodies (IgG) was raised against the haemagglutinin of an early representative of the Hong Kong (H3N2) subtype of influenza. They were classified into groups based on their cross-reactions with 16 other virus strains from the same subtype. This classification was performed using methods of numerical taxonomy. Statistical tests supported the validity of the grouping. Ten such groups were identified. Nine antibodies remained unclassified. The locations on the haemagglutinin molecule of amino acid residues influencing the binding of each antibody group were estimated. This was achieved by a study of antibody cross-reactive profiles, coupled with previously published locations of amino acid changes in the primary sequence of different haemagglutinins, and their positions in the tertiary structure of the molecule. The locations of the amino acids affecting antibody binding overlapped between the different antibody groups forming a continuous ring surrounding the probable cell-receptor pocket. The amino acids affecting the binding of each antibody group may or may not represent the actual antibody binding sites. The importance of the different sites of amino acid variation in the haemagglutinin during evolution of the virus is discussed.

Virus isolation and serological studies on swine sera collected during 1973 to 1978 showed that H1N1 (Hsw1N1) influenza viruses first appeared in the swine population of Japan about May 1977. With the exception of one strain, both haemagglutinin and neuraminidase subunits of all the H1N1 viruses isolated from swine in Japan and from pigs imported from North America were antigenically indistinguishable from those of A/NJ/8/76 virus, suggesting the introduction of swine influenza virus into Japan with imported pigs from North America as breeding stock. Antigenic analysis of a recombinant virus by neuraminidase-inhibition tests with specific antisera to the isolated neuraminidases of A/Victoria/3/75 and A/Aichi/2/68 revealed that the neuraminidase antigen of the recombinant virus, A/swine/Kanagawa/2/78 (H1N2), was closely related to those of A/Tokyo/6/73 (H3N2) and A/Kumamoto/22/76 (H3N2) viruses.

We have identified the cap-recognizing protein of two strains of influenza A fowl plague virus (FPV) by photoaffinity labelling of virion proteins with a photoreactive analogue of the 5’-methyl cap structure of messenger RNA. The cap-recognizing protein of influenza A/FPV/Rostock/34 is the P2 polypeptide, and that of influenza A/FPV/Dutch/27 (Dobson) is the P3 polypeptide. In each case the cap-recognizing protein is the product of RNA segment 1.

An immunoradiometric assay for human interferon-α (HuIFN-α) has been adapted for the assay of low concentrations of HuIFN-α in human serum. The sensitivity of the assay is 5 to 10 IU/ml and the coefficient of variation less than 10%. The assay was shown to compare well with a biological (antiviral) assay in the measurement of serum interferon following intramuscular injection of HuIFN-α in nine volunteers. Serum interferon was also measured in the serum from 250 normal human donors. Two donors appeared to have detectable levels of HuIFN-α.

Evidence ispresented for the presence of a covalently linked protein that isattached to the 5'-ends of genomic RNA of pea enation mosaicvirus ( PEMV). Nuclease digestion of PEMV RNA released protein with anapparent mol. wt. of approx. 17 500. The absence of a 5'-capand the inability to introduce [32P]phosphate into the5'-termini of these RNAs suggests that the protein iscovalently bound to PEMV RNA.