Genetics of reovirus: the relationship of interference to complementation and reassortment of temperature-sensitive mutants at nonpermissive temperature.

Temperature-sensitive mutants of reovirus type 3 are capable of interfering with the replication of wild-type reovirus type 3. The interfering activity correlated with the ability of pairs of mutants to complement at 39°: Pairs of noninterfering mutants (tsD × tsE) yielded efficient complementation (indexes of 10–50); pairs of interfering mutants (including members of groups ts A, B, G) did not produce significant complementation (indexes ～ 1). The ability of pairs of mutants to reassort at 39° generally followed a similar pattern. Thus interference is an important property of ts mutants of reovirus and needs to be considered when genetic interactions are being studied at 39°.     

A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D, and E to genome segments

The double-stranded RNA (dsRNA) of recombinants derived from crosses of the dsRNA-negative, temperature-sensitve (ts) mutants of reovirus type 3 and reovirus serotypes 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments containing the ts lesions. In this way the location of the mutation of the group C prototype mutant tsC(447) os genome segment S2, that of the group D prototype mutant tsD(357) is genome segment L1, and that of the group E prototype mutant tsE(320) is genome segment S3. In addition the location of the temperature-sensitive lesion of serotype 2 is genome segment S1.     

Genetics of reovirus: Identification of the ds RNA segments encoding the polypeptides of the μ and σ size classes

Recombinants derived from crosses between reovirus serotypes 1, 2, and 3 were utilized to identify the ds RNA segments encoding polypeptides of the μ and σ size classes. The results indicate the following assignments: M1 ds RNA encodes polypeptide μ2, M2 encodes μ1, M3 encodes μNS, S1 encodes σ1, S2 encodes σ2, S3 encodes σNS, and S4 encodes σ3. In addition polypeptide species μ2 has been conclusively identified as a structural component of the reovirus core. A new nomenclature for the viral polypeptides is discussed.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

Intercistronic complementation between adenovirus 2 temperature-sensitive mutants.

The mechanism of complementation, dominance, and the effects of gene dosage were examined by means of sodium dodecyl sulfate-polyacrylamide gel analysis of infected cell proteins and purified virions synthesized in cells coinfected with adenovirus type 2 temperature-sensitive mutants. In particular the synthesis of a 50K core-polypeptide, related to protein V, and the processing of core protein PVII were examined. The efficiency of complementation was unique to each mutant. Coinfection with wild-type failed to suppress completely the mutant phenotype suggesting that the mutants were defective in non-catalytic proteins. Interserotype complementation between Ad2ts3, a hexon mutant, and Ad5ts22, a fiber mutant, resulted in mosaic virions with Ad2 fibers and Ad5 hexons, rather than in mosaic capsomeres. The putative hexon mutant, ts3, was found to exert a gene frequency-dependent dominance. Evidence was also obtained suggesting that the ts3-specific 50K polypeptide may be a precursor to core protein V. We conclude that complementation is profoundly influenced by the nature of the participating gene products and their function in virus infection.     

Replication of simian virus 40 in simian virus 40-transformed hamster kidney cells induced by mitomycin C or 60Co gamma irradiation.

Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with ⁶⁰Co γ irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluoresence techniques, respectively. Only some cells in induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or ⁶⁰Co γ irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of γ irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication.     

A genetic map of reovirus. III. Assignment of the double-stranded RNA-positive mutant groups A, B, and G to genome segments

The double-stranded RNAs (dsRNA) of recombinants of the RNA-positive, temperature-sensitive (ts) mutants of reovirus type 3 and reovirus serotype 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments responsible for the ts lesions. In this way the location of the mutation of the group A prototype mutant tsA(201) is genome segment M2, that of the group B prototype mutant tsB(352) is genome segment L2, and that of the group G prototype mutant tsG(453) is genome segment S4.     

Immunological evidence for the synthesis of all canine distemper virus polypeptides in chronic neurological diseases in dogs. Chronic distemper and old dog encephalitis differ from SSPE in man.

Immunoprecipitation of the polypeptides of canine distemper virus (CDV) from lysates of infected cells has revealed that the serum and cerebrospinal fluid (CSF) of dogs with chronic distemper and old dog encephalitis contain high levels of antibody to all the viral structural polypeptides, indicating that all of these polypeptides are being synthesized in the dogs. These findings, and the fact that infectious virus has been isolated from explants of brain tissue without cocultivation techniques, differ from those with measles virus in subacute sclerosing panencephalitis (SSPE) in which there is a lack of antibody to the virus membrane (M) protein and cocultivation of brain explants with permissive cells is required for virus isolation. These results indicate that CDV undergoes complete replication in the brain in the persistent infection resulting in chronic neurological disease, in contrast to the situation with measles virus in SSPE in which replication appears to be abortive.     

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.     

Sendai virus hemolysis: influence of lectins and analysis by immune fluorescence.

Hemolysis by Sendai virus has been assayed in a system of erythrocytes attached in monolayer fashion to plastic surfaces by poly-l-lysine. Immobilization of cells in this way allowed the easy performance of multiple-step experiments in which the effect of virus was studied in conjunction with lectins and antibodies. The binding of these ligands immediately prior to or after the adsorption of virus particles to erythrocyte monolayers had a marked inhibitory effect on viral hemolysis. Different patterns of hemolysis resulted according to the specificity of the lectins and antibodies with respect to the virus receptor. Con A, Ricinus communis agglutinin, and anti-Sendai antibodies, reacting with receptors distinct from the sialoglycoprotein, did not interfere with the adsorption process of the virus to the membrane. They inhibited hemolysis exclusively by a restriction of the mobility of elements in the cell membrane and in the viral envelope. Phytohemagglutinin, wheat germ agglutinin, and anti-glycophorin antibodies, on the other hand, inhibited lysis by interference with elements including the virus receptor. It is concluded that the induction of hemolysis requires the specific binding of the virus to a receptor and that rearrangement of elements in both the viral envelope and the plasma membrane have to follow this adsorption. These subtle molecular rearrangements are a prelude to virus-cell fusion in which disintegration of the viral envelope structure is observed. This latter process could be characterized by monitoring the expression of virus and/or host-specific antigens with immune fluorescence techniques. Reactions of virus-bound erythrocytes with anti-host did not interfere to a detectable degree with the subsequent binding of anti-viral antibodies. In contrast, antibodies directed against virus-specific antigens and attached to virus-bound erythrocytes at 4° but not at 37° inhibited the subsequent reaction of virus with anti-host. We conclude that two types of host antigens exist: those accessible in intact virus particles and closely associated with the distal portion of those viral proteins forming spikes, and those antigens reactive only after the dispersal of the elements of the viral envelope folowing its fusion with the cell surface.     

Proteins of murine cytomegalovirus: identification of structural and nonstructural antigens in infected cells.

The synthesis of viral proteins in tertiary mouse embryo cells infected with murine cytomegalovirus (MCMV) has been studied by polyacrylamide gel electrophoresis. Three immediate-early proteins were detected by 4 hr postinfection (p.i.), but between 4 hr and the onset of viral DNA synthesis at 8–10 hr, no further viral proteins could be distinguished. The major structural proteins appeared after viral DNA synthesis had commenced and continued to be made until 36 hr p.i. or later. Host translation occurred until 24 hr p.i. but was inhibited between then and 30 hr p.i. at which time viral protein synthesis predominated. At this time 52 proteins could be labeled in infected cells and, of these, 30 could be precipitated with viral-specific antisera. The proteins precipitated included 22 with electrophoretic mobilities similar to structural viral proteins and a further 8 which were not present in purified MCMV. These, together with the three immediate-early proteins, were the only nonstructural proteins which were detected in the infected cell.     

A new method for titration of murine leukemia virus using purothionin A.

A basic polypeptide, purothionin A, which selectively injures cells in the S phase was tested on A31 cells producing MuLV. Treatment with 10 μg/ml PA has a marked cytotoxic effect on contact inhibited A31 cells infected with Moloney murine leukemia virus (Mo-MuLV), but scarcely any effect on uninfected A31 cells. On the basis of this finding we developed a new method for titration of MuLV: Cultures infected with highly diluted MuLV, which showed no XC plaques, were not affected appreciably by purothionin A, whereas culture infected with low dilutions of MuLV, in which large numbers of XC plaques normally developed, were severely affected by purothionin A. The results of this assay method were consistent with those obtained by XC plaque assay.     

The effect of verapamil on myocardial ultrastructure during and following release of coronary artery occlusion

Although the calcium channel blocking agent verapamil has been shown to have beneficial effects on ischemic myocardium, its effect on cardiac ultrastructure during regional myocardial ischemia and following coronary reperfusion has not been studied in detail. The purpose of this study was to investigate the effect of verapamil on the ultrastructure of myocardium during the early phase of ischemia and following coronary reperfusion. Open-chest anesthetized dogs were subjected to 1 hr of occlusion of the proximal left anterior descending coronary artery followed by 1 hr of reperfusion. The mean ischemic score, a semiquantitative index of ultrastructural damage, was significantly lower in verapamil-treated (0.9 ± 0.3) than in untreated dogs (1.9 ± 0.2, P < 0.025) during coronary occlusion and especially following reperfusion (1.0 ± 0.3 versus 2.9 ± 0.5, respectively P < 0.025). Verapamil treatment prevented the hastening of ultrastructural damage (explosive cell swelling phenomenon) associated with reperfusion into severely ischemic myocardium. Verapamil resulted in a more profound reduction of the extent of ultrastructural damage of mitochondria compared to other organelles. Thus, this study supports the concepts that verapamil reduces ultrastructural damage both during coronary occlusion and following coronary reperfusion and may reduce ischemic damage by protecting mitochondrial structure.     

Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins.

We have employed monospecific antisera to the major glycoproteins (gp52 and gp36) and the major nonglycoprotein (p27) of the mouse mammary tumor virus (MMTV), and we now report the first isolation of an intracellular MMTV precursor polypeptide to p27. The precursor polypeptide to p27 (Pr75) binds to single-stranded DNA (ssDNA) and can be easily separated from the precursor to gp52 and gp36 (gPr75) by ssDNA-Sepharose column chromatography. [³⁵S]Methionine-labeled Pr75 contained tryptic peptides of p27 and p14 of MMTV. Protein p14 has previously been shown to be capable of binding to ssDNA. In contrast, [³⁵S]methionine-labeled gPr75 contained tryptic peptides of only gp52 and gp36, neither of which binds to ssDNA.     

DNA from two Orgyia pseudotsugata baculoviruses: molecular weight determination by means of electron microscopy and restriction endonuclease analysis.

Both aqueous and urea-formamide procedures for spreading nucleic acid were employed for electron microscopic studies on the DNAs from the nucleopolyhedrosis bundle virus (NPBV) and the nucleopolyhedrosis single-rod virus (NPSV) both of which are pathogenic for Orgyia pseudotsugata. The molecular weight estimates via electron microscopy were derived by comparison of the mean length values for the double-stranded relaxed circular DNAs with that of SV40 DNA. The aqueous and urea-formamide spreading methods yielded NPSV DNA molecular weight values of 103 × 10⁶ and 104 × 10⁶ daltons, respectively, and molecular weight values of 87 × 10⁶ and 85 × 10⁶ daltons for NPBV DNA. These molecular weights were compared with molecular weight estimates from restriction endonuclease analysis, sedimentation analysis, and renaturation kinetic analysis. DNA located within the NPBV polyhedra but external to virions was characterized by restriction endonuclease analysis and examined by electron microscopy. It was determined to be composed of fragments of random size and to be viral origin.     

RNA-binding properties of nonstructural polypeptide G of encephalomyocarditis virus.

Extracts of encephalomyocarditis (EMC) virus-infected Krebs II cells were subjected to chromatography on either EMC virus RNA or different homopolyribonucleotides immobilized on cellulose. Among several EMC virus-specific proteins, the nonstructural polypeptide G was shown to bind selectively to RNA. This binding appears to be relatively unspecific with respect to nucleotide sequence.     

Translation of PVX RNA in vitro by wheat germ. I. Characterization of the reaction and product size

Potato virus X (PVX) RNA was an efficient mRNA for in vitro translation by the wheat germ system. Optimal incorporation of labeled amino acids occurred in reaction mixtures (50 μl) containing 3 mM Mg²⁺, 70 mM K⁺, and 3–4 μg of RNA. The presence of PVX RNA stimulated amino acid incorporation up to 40 times over background levels. Addition of spermine or spermidine further stimulated amino acid incorporation nearly twofold. PVX RNA translation was not inhibited by adding double-stranded RNA from Penicillium stoloniferum. The largest of several polypeptides obtained from PVX RNA translation reaction mixtures had an apparent molecular mass of 110,000 daltons. No polypeptide that coelectrophoresed with native PVX coat protein subunits was observed even when heat-treated PVX RNA was used, or when the products from spermine-stimulated reactions were analyzed.     

The genes coding for the surface glycoproteins of influenza A viruses contain a small conserved and a large variable region.

The variable genes of influenza A viruses coding for hemagglutinin and neuraminidase consist of a relatively small highly conserved region, which is presumably involved in the functional integrity of the gene products, and a relatively large highly variable region, presumably involved in the immunological properties. This is demonstrated by melting profiles of homologous and heterologous RNA hybrid molecules. The results are discussed as a possible mechanism how serologically different influenza strains evolve by mutation in the variable part and selection by the immune system of the host.     

Studies on the generation and amplification of sendai virus defective-interfering genomes.

Sendai virus which had been cloned by successive plaque purification was found to generate a wide assortment of defective-interfering (DI) genomes on continued high-m.o.i. passage in eggs. No predisposition to generate a particular DI genome was noted. One stock of Sendai virus, which contained 13 different DI genomes varying in length from 670 to 7100 nucleotides was passaged undiluted in eggs for several more generations to see whether the smaller DI genomes could be selectively amplified. Our results indicate that the size of the DI genome per se does not confer a selective advantage and the implications of this finding are discussed.