- Volume 64, Issue 1, 1983

Rapidly sedimenting complexes (RSCs) of replicating phage K DNA, isolated by rate zonal centrifugation in sucrose gradients, contain bacterial membrane lipids and protein. During the first half of the latent period the number of DNA molecules in a RSC increased from 1 to about 27. Digestion by Pronase caused the complexes to dissociate and release virion lengths of DNA which sedimented slowly like free mature DNA. RSCs treated with SDS disintegrated and released tangled DNA molecules, each about one virion length in size, but these structures retained their fast sedimentation characteristic. Chloramphenicol (CM) at 100 µg/ml did not completely inhibit complex formation or DNA replication, indicating that pre-existing host proteins were involved in these processes. CM reduced DNA replication by 50 to 80%. It is concluded that phage K DNA replicates attached to the cytoplasmic membrane of the host.

Hybrids between Escherichia coli phage ϕ80 and Salmonella typhimurium phage P22 were isolated after superinfection by P22 of a smooth E. coli-S. typhimurium hybrid lysogenic for ϕ80. These hybrid phages, designated ϕ80immP22 and ϕ80immP22dis, possessed the ϕ80 protein coat and tail genes. The ϕ80immP22 hybrids acquired the immunity (immC) region of P22 and some adjacent P22 genes, but E. coli-S. typhimurium strains lysogenic for ϕ80immP22 hybrids remained sensitive to P22. The ϕ80immP22dis hybrids, found ten times more frequently than the ϕ80immP22 hybrids, contained a more extensive portion of the P22 genome which encompassed the immI as well as the immC region of P22. Therefore, the ϕ80immP22dis hybrids conferred on their hosts immunity to P22 infection. Further analyses have revealed that the ϕ80immP22dis hybrids carry the P22 attachment region and either P22 tail gene 9 or antigen conversion gene al, but not both of these genes.

We have continued our studies on the effect of high temperature (42 °C) on the development of RNA bacteriophage MS2 in the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39 carrying a traD (ts) mutation. At 42 °C, mutant cells permit the penetration and translation of phage MS2 RNA but do not permit MS2 RNA replication. We suggest a role for the traD (ts) mutation in MS2 RNA replication.

The synthesis and accumulation of more than 30 virus-induced polypeptides was detected after infection of permissive primate cells with high multiplicities of herpesvirus saimiri. These virus-induced polypeptides had apparent mol. wt. of from 12000 (12K) to 250K and differed in molar abundance by up to two orders of magnitude. The majority of virus-induced polypeptides satisfied multiple criteria for their virus specificity. Polypeptides of 110K, 76K, 51K and 31K to 29K were synthesized at maximum rates early in infection, but the majority of proteins were made at high rates late in infection. Virus-specific polypeptides were substrates for a number of post-synthetic modifications. The 117K, 85K, 76K, 31K and 30K polypeptides were each processed to forms with altered electrophoretic mobility. The 117K and 85K polypeptides were among the virus-specified substrates for glycosylation and virus-induced polypeptides of 59K, 51K, 30K and 26K were phosphorylated. The early 51K phosphoprotein and the 30K to 31K polypeptides were rapidly translocated to the nucleus of infected cells. The 31K polypeptide was processed to a 29K product which remained stably associated with the nuclear fraction. The 30K nuclear protein was shown to be the precursor of a 28K polypeptide which was released in a soluble form into the cytoplasmic fraction and the culture medium of cells at late times in the virus growth cycle. Many other polypeptides accumulated slowly in nuclear (e.g. 250K, 150K, 130K, 110K and 38K) or in cytoplasmic (e.g. 117K, 85K and 28K) fractions of infected cells in forms which could be differentiated by the use of detergents or differences in stability to salt extraction. The mol. wt., relative molarities and some features of the post-synthetic processing of herpesvirus saimiri polypeptides more closely resembled the properties of gene products of Epstein—Barr virus than those of herpes simplex virus.

The addition of TPA (phorbol-12-myristate-13-acetate) to cultures during the lytic infection with herpesvirus saimiri led to an enhanced and accelerated production of polypeptides induced by H. saimiri and to a rapid shut-down of host cell protein synthesis and allowed a detailed analysis of the protein patterns. Analysis of sequential protein synthesis in owl monkey kidney cells lytically infected with H. saimiri 11 permitted the identification of 31 virus-induced polypeptides. The use of the amino acid analogues canavanine (for arginine) and azetidine (for proline) in parallel allowed experiments on the identification of proteins synthesized early and late during lytic infection.

The physical maps of the DNAs of two cytomegalovirus isolates, AD169 and SG, were compared by cross-blot hybridization and by hybridization of nitrocellulose-bound SG fragments with cloned 32P-labelled AD169 fragments. From this comparison it can be concluded that both physical maps are co-linear to a large extent. Most variability existed at the termini of the long and short component (at the repeats). Other differences were the presence or absence of restriction endonuclease cleavage sites.

Nude mice (nu/nu), heterotransplanted with human tumours and kept in isolators, were found to suffer from wasting and posterior paralysis. Electron microscopy of spinal cord tissue revealed virus particles in the oligodendrocytes consistent in size (35 to 40 nm), morphology and distribution with those of the polyoma-SV40 sub-group of papovaviruses. Serology and restriction enzyme analysis of the virus genome showed that the virus was the murine polyoma A2 strain. Inoculation of uninfected nude mice with 107 TCID50 of polyoma A2 strain virus produced a similar disease in these mice with wasting and, after 10 to 23 weeks, paralysis of the hind legs of all surviving mice. Extensive myelin disruption was seen throughout the brain stem and sacral region of the spinal cord and high titres of polyoma virus were found in the whole brain (108.8 TCID50/brain) and in the spinal cord (106.8 TCID50/spinal cord).

In order to find out whether polyoma virus (Py) integration into chromosomes causes rearrangements in the cell DNA flanking the integration site, we have mapped the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts, and then compared the two maps. To carry out this study we have cloned a segment including Py DNA and flanking sequences in the bacteriophage vector λgtWES and subcloned the flanking cell DNA in a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes and used the cloned flanking cell DNA and Py DNA as hybridization probes. Autoradiography of the LPT DNA blots revealed two sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the second set consists of fragments which contain no virus DNA sequences, and are identical to the fragments observed in the corresponding normal rat DNA digests. These data indicate that LPT cells are heterozygous with respect to the Py inserts. The same data were used to map the flanking sequences in the two types of cells. A comparison between the two maps revealed that a 3.0 kb cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, has been deleted from the LPT chromosome which carries Py DNA, but not from the LPT chromosome which does not carry the virus DNA. The implications for papovavirus integration are discussed.

Virus gene expression in rat cells transformed by either avian myelocytomatosis virus strain MC29 or avian erythroblastosis virus has been studied by biological and biochemical methods. In the clones examined, virus-specific sequences were found to be transcribed into RNA and, in most clones, the characteristic gag-related proteins could be identified. The transformed rat cells were fused to permissive chick cells and the rescued virus was shown to transform both chick embryo fibroblasts and the appropriate haemopoietic cell type in chick bone marrow cultures. These results clearly demonstrate that, as with the non-defective avian sarcoma viruses, the genetic information responsible for transformation by the defective avian leukaemia viruses can be expressed in non-permissive mammalian host cells as well as in permissive avian cells.

We have recently found that, in vitro, the murine leukaemia virus (MuLV)-associated protein kinase activity predominantly phosphorylates Pr65gag, a virus protein present in relatively small amounts in partially purified virus preparations. Other virus proteins, such as p10, Pr27gag and Pr40gag, are also phosphorylated in vitro, but to a lesser degree. Furthermore, when immature core subparticles which are enriched in Pr65gag are prepared from virions by Sepharose 6B exclusion column chromatography, about 50% of the kinase activity (as assayed with the exogenous substrate phosvitin) remains associated with the cores. We report here that this core-associated activity is distinct from Pr65gag since it can be separated from Pr65gag by chromatography on denatured DNA-cellulose columns followed by centrifugation of the 0.2 m-NaCl-eluted fraction. Under these conditions, Pr65gag is pelleted while the kinase activity, which can phosphorylate both endogenous (MuLV Pr65gag and p10) as well as exogenous (phosvitin) substrates, remains in the supernatant. Interestingly, when the amount of Pr65gag is reduced, as in such preparations, p10 then becomes more heavily phosphorylated.

The multiplication of murine coronavirus strains A59 or JHM in Sac(-) cells was unaffected by the presence of α-amanitin at concentrations which inhibited the host cell DNA-dependent RNA polymerase activity. In cells infected with the A59 virus strain, actinomycin D-resistant RNA synthesis could readily be detected by pulse-labelling with [3H]uridine; this virus-specific RNA synthesis was not induced in the presence of the protein synthesis inhibitor anisomycin. A new RNA-dependent RNA polymerase activity was detected in the large particle fraction of A59 virus-infected cells. Optimal conditions for enzyme activity in vitro were established. Maximum activity occurred 5 h after infection, coincident with the peak of virus-specific RNA synthesis detected by pulse-labelling in vivo.

Protein synthesis in the murine hepatitis virus JHM-infected cells was temporarily inhibited by hypertonic shock. When the cells were returned to isotonic medium the synthesis of six virus-specific polypeptides, 150K, 65K, 60K, 30K, 23K and 14K was reinitiated simultaneously. Polyadenylated RNA isolated from the cytoplasm or polysomes of infected cells was translated in vitro and the products included polypeptides with molecular weights (mol. wt.) of 120000, 60000, 30000, 23000 and 14000. Immunoprecipitation and fingerprinting of [35S]methionine-containing tryptic peptides showed that the 60000 and 23000 mol. wt. products were identical to the 60K and 23K polypeptides found in infected cells; the 120000 mol. wt. product showed identity with the 150K intracellular polypeptide and a virus-specific 120K polypeptide synthesized in tunicamycin-treated cells. Two-dimensional polyacrylamide gel electrophoresis strongly suggested that the 30000 and 14000 mol. wt. products are equivalent to virus-specific 30K and 14K intracellular polypeptides. [3H]Uridine-labelled polyadenylated virus RNA was isolated from infected cells and sedimented in sucrose gradients containing formamide. The distribution in the gradient of each of the previously identified virus RNAs was determined by gel electrophoresis and gradient fractions enriched for each RNA were translated in vitro. The 120000, 60000, 30000, 23000 and 14000 mol. wt. polypeptides were found to be encoded by mRNAs 3, 7, 2, 6, and 4 or 5 respectively. These results demonstrate that the virus-specific polypeptides in JHM-infected cells are encoded in separate subgenomic mRNAs and are translated independently. The assignment of coding functions and the known sequence relationships of JHM RNAs permitted a gene order to be deduced.

Genome RNA of mouse hepatitis virus (MHV) strain A59 has been used as a template to synthesize two virus-specific probes: cDNArep, representing the majority of sequences of the genome RNA and cDNA3′, representing the 3′ end of the genome RNA. Molecular hybridization with these cDNAs was used to characterize both genome RNA and intracellular virus-specific RNAs. Hybridization of genome RNAs of MHV strains A59, JHM, and MHV-3 with A59 cDNArep showed that, although these three strains exhibit different pathogenicities, they contain closely related nucleotide sequences. Hybridization of intracellular RNA from MHV-infected cells with virus-specific cDNA shows that (i) the majority of virus-specific RNA is polyadenylated, (ii) virus-specific intracellular RNA contains six subgenomic species of the same polarity as genome RNA and (iii) all subgenomic RNAs contain the same 3′ sequences as the genome RNA and thus form a nested set of RNAs.

Unclassified Venezuelan equine encephalitis (VEE) viruses Tonate (TON), Bijou Bridge (BB), Paramana (PARA), 71D-1252 and Cabassou (CAB) were characterized serologically and biochemically. The envelope glycoproteins of these and nine other VEE viruses representing VEE subtype variants I-AB, I-C, I-D, I-E, II, III and IV were separated by column isoelectric focusing. The E1 and E2 glycoproteins of all the Zwittergent-dissociated VEE viruses focused at pI 6.3 to 6.9 and pI 8.6 to 9.3 respectively. Haemagglutination-inhibition and neutralization tests using rabbit sera to the E2 glycoprotein of TON, BB and PARA viruses showed them to be indistinguishable from each other and closely related to prototype subtype III virus Mucambo (MUC). VEE strain 71D-1252 was also serologically closely related to prototype MUC virus. We proposed that MUC, TON and 71D-1252 VEE viruses be classified subtype III viruses, designated variants III-A, III-B and III-C respectively. CAB virus, which is not closely related to other VEE isolates, may represent a new VEE subtype (V). SDS-PAGE resolved the capsid protein (35 to 36 kdal) and two major envelope glycoproteins of 50 to 51 kdal (E1) and 51 to 58 kdal (E2) for all VEE viruses except CAB; the two glycoproteins of CAB virus co-migrated by PAGE with apparent identical mol. wt. of 51 kdal. Limited digestion of SDS-dissociated virus proteins with Staphylococcus aureus V8 protease produced identical peptide maps for serologically indistinguishable viruses. Oligonucleotide fingerprinting of virus RNA supported the close serological relationships observed at the genome level.

The first step in virus replication is attachment of the virus to the host cell surface. To investigate this process, the binding of TC-83 (the attenuated strain of Venezuelan equine encephalomyelitis virus) to BW-J-M (a macrophage-like murine cell culture line) has been characterized. The binding of radiolabelled virus can be blocked by excess unlabelled virus and has a pH optimum in the physiological range. Binding is saturable; analysis using Scatchard plots or the computerized binding data analysis program, LIGAND, yielded estimates of a single class of 4 × 105 binding sites per cell and an equilibrium binding constant of 2.0 ± 0.7 × 1012 m −1. Virus bound to the cell could be visualized by transmission electron microscopy and was localized primarily in coated regions of the membrane. Virus, bound under optimum conditions at 0 °C, was internalized upon warming and infected cells productively. Treatment of BW-J-M cells with low concentrations (1 to 10 µg/ml) of the proteolytic enzymes trypsin, Pronase or proteinase K caused a dose-dependent reduction in binding capacity. Trypsin-treated cells, upon return to culture, progressively regained their binding capacity within 24 h. As a further characterization of the virus binding site, several lectins were studied for their ability to inhibit TC-83 binding to BW-J-M cells. Canavalia ensiformis agglutinin, Glycine max agglutinin and Triticum vulgaris agglutinin were potent inhibitors of virus binding. This evidence suggests that TC-83 binds to a specific receptor on the BW-J-M cell and that the receptor may be glycoprotein.

The specificity of the IgM response in acute human coxsackievirus B infections was examined by indirect solid phase enzyme immunoassay and immunoblot techniques. IgM antibodies detected by ELISA were either strictly type-specific, type-predominant or group-reactive to coxsackieviruses B-1 to B-5. Homotypic and type-dominant responses were clearly correlated with the serotype isolated from the patient. Analysis of ELISA antigens by SDS-gel electrophoresis, protein transfer and subsequent immunodetection of individual virus polypeptides, revealed VP1 as the major antigen; it was detected by homotypic as well as heterotypic serum specimens.

Serological studies (complement-fixation, immunofluorescent and plaque reduction neutralization) were carried out with 50 vertebrate rhabdoviruses to determine their antigenic relationships. On the basis of these studies, an antigenic classification of rhabdoviruses infecting terrestrial vertebrates is proposed. This classification includes three major and four minor serogroups. Several new members of the vesicular stomatitis serogroup were identified.

Fifteen strains of influenza C virus were isolated from abattoir pigs in China in 1981 and antibody against influenza C virus was found in pig sera. Virus survey also showed seasonal activity of influenza C virus in pigs. Additionally, experimental infection of pigs with influenza C virus demonstrated that Chinese domestic pigs could be infected by influenza C virus and that the virus could be transmitted from pig to pig. These results indicate that, like influenza A, influenza C virus can cause natural infection in pigs. To our knowledge, this is the first report of such a finding.