bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc-lesion
Authors: Chartrand P , Timbury M C , Hay J , Moss H
Published: January 2020

We had previously shown that a temperature-sensitive (ts) mutant of herpes simplex virus type 2 strain HG52, ts13, induced a heat-labile DNase activity in infected cells (B. Francke, H. Moss, M. C. Timbury, and J. Hay, J. Virol. 26:209-213, 1978). Earlier work indicated that the mutant also possessed temperature-sensitive infectivity (I. W. Halliburton and M. C. Timbury, J. Gen. Virol. 30:207-221, 1976). In this study temperature-stable revertants of ts13 have been isolated; examination of them revealed that ts13 is a double mutant, with genetically distinct temperature-sensitive lesions affecting nuclease activity and particle stability. The lethal mutation, in the cell system studied, is the latter. Revertants, which all maintain the nuclease lesion, grew well at a high temperature. Physical mapping of the nuclease lesion placed it between 0.12 and 0.21 (fractional length) on the virus genome, quite distant from the lethal mutation at 0.64 to 0.70.
Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses
Authors: Keränen S , Kääriäinen L
Published: January 2020

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.
Abnormal properties of an immediate early polypeptide in cells infected with the herpes simplex virus type 1 mutant tsK
Authors: Preston C M
Published: January 2020

Previous studies (R. J. Watson and J. B. Clements, Virology 91:364--379, 1978; C. M. Preston, J. Virol. 29:275--284, 1979) have shown that the herpes simplex virus type 1 (HSV-1) mutant tsK has a temperature-sensitive lesion in an immediate early polypeptide whose function is to induce synthesis of new viral transcripts, including mRNA, for pyrimidine deoxyribonucleoside kinase. The studies presented here examine the properties of immediate early polypeptides in wild-type HSV-1 and tsK-infected cells at 31 and 38.5 degrees C. The overall pattern of immediate early protein synthesis was similar in wild-type HSV-1- and tsK-infected cells when radiolabeled with [35S]methionine or 14C-amino acid mixture. Further investigation, however, revealed two aberrant properties of the polypeptide Vmw 175 in tsK-infected cells at 38.5 degrees C. Upon cell fractionation, large amounts of this polypeptide were recovered in the cytoplasmic fraction, in contrast to tsK-infected cells at 31 degrees C or wild-type HSV-1-infected cells at either temperature. Furthermore, at 38.5 degrees C tsK-induced Vmw 175 was not processed normally to forms of lower electrophoretic mobility. Both of these defects were reversible upon downshift of tsK-infected cells, even in the absence of further protein synthesis, but were not observed in cells infected with a revertant of tsK. Coinfection of tsK-infected cells with wild-type HSV-1 did not alleviate these lesions, suggesting that they resulted from an abnormal Vmw 175 polypeptide rather than from a defective processing enzyme. Temperature upshift of tsK-infected cells caused reversion of Vmw 175 to the mutant form. The progression to synthesis of late polypeptides was also arrested; therefore, a functional lesion was also reversible upon temperature changes between 31 and 38.5 degrees C during the early stages of infection. The identification of a polypeptide with abnormal properties in tsK-infected cells and the demonstration that these properties, and the functional lesion, are reversible may provide an important system for investigation of HSV-1 transcriptional control.
Inhibition of Rous sarcoma virus replication by 2-deoxyglucose and tunicamycin: identification of an unglycosylated env gene product
Authors: Stohrer R , Hunter E
Published: January 2020

Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.
DNase Induced After Infection of KB Cells by Herpes Simplex Virus Type 1 or Type 2 II. Characterization of an Associated Endonuclease Activity
Authors: Hoffmann Peter J. , Cheng Yung-Chi
Published: January 2020

Purified preparations of the “exonuclease” specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg
2+
or Mn
2+
is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca
2+
, whereas no activity occurs in the presence of Zn
2+
. In the presence of Mg
2+
, Ca
2+
and Zn
2+
are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn
2+
greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg
2+
. The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg
2+
than Mn
2+
presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.
Salmonella bacteriophage glycanases: endorhamnosidases of Salmonella typhimurium bacteriophages
Authors: Svenson S B , Lönngren J , Carlin N , Lindberg A A
Published: January 2020

Twelve bacteriphages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology--an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off [14C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the alpha-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the alpha-L-rhamnopyranosyl-(1 leads to 3)-D-galactopyranosyl linkage in the S. typhimurium O-polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the O-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and D1, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6-dideoxyhexopyranosyl group (R). : FORMULA:(SEE TEXT). The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at O-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at O-4 of the D-galactopyranosyl residues, the adjacent alpha-L-rhamnopyranosyl linkages were found to be perferentially cleaved.
Electrophoretic characterization of intracellular forms of bacteriophage phi X174 DNA: identification of novel intermediate of altered superhelix density
Authors: Johnson P H , Miller M J , Wild E , Kelly S V , Grossman L I
Published: January 2020

The replication cycle of bacteriophage phi X174 DNA has been analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and40 min after infection was deduced and quantitated. Migration through 1.4% agarose at 5 and 10 V/cm resolved all known viral DNA species as well as fragments of host chromosomal DNA. Among parental replicative form(RF) molecules synthesized, 1 to 3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most of the input viral strands remained in a duplex structure throughout the period of infection studied here. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF were found to exist as form I dna. in addition, approximately 1% of the viral DNA was found as unit length linear single strands. Electrophoretic analysis of RF DNA after controlled denaturation suggests the existence of four populations of closed circular RF: (i) molecules of native superhelix density (RFI); (ii) a population of molecules of altered topological linking number, alpha, differing in increments of one superhelical turn (tau) between tau values of 0 and approximately -31; (iii) a superimposed population of topological isomers which under electrophoresis conditions have mean tau value (tau) equal to +5; and (iv) a population of "complexed" molecules with a reduced number of superhelical turns due to their association with single-stranded DNA and RNA. Complexed parental molecules isolated from cells infected at high multiplicity released FRI and homologous single-stranded DNA upon denaturation and are postulated to be intermediates in genetic recombination. Complexed RF DNA isolated from cells infected at low multiplicity release native supercoils upon reaction with RNase H and are observed by electron microscopy to contain displacement loops. Such molecules are likely intermediates in transcription. Our results are consistent with a structure of complexed RFI involving a partially triple-stranded helix in which a covalently closed circular duplex molecule contains a reduced number of superhelical turns due to the unwinding produced by base pairing between one strand of the supercoil and an associated homologous single strand of DNA or RNA.
Defective Influenza Viral Ribonucleoproteins Cause Interference
Authors: Janda J. Michael , Nayak Debi P.
Published: January 2020

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [
32
P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.
The nonstructural component of the Abelson murine leukemia virus polyprotein P120 is encoded by newly acquired genetic sequences
Authors: Van de Ven W J , Reynolds F H , Nalewaik R P , Stephenson J R
Published: January 2020

The Abelson leukemia virus (AbLV) polyprotein P120 is compared to translational products representing the entire Moloney murine leukemia virus (MuLV) genome on the basis of [35S]methionine tryptic peptide composition. Three methionine-containing tryptic peptides present in Moloney Pr65gag are each shown to be present in both Pr75gag and in Pr180gag-pol. Of these, one peptide, corresponding to Moloney MuLV p12, but neither of two p30-specific peptides are present in AbLV P120. Among the 12 remaining methionine-containing peptides present in AbLV P120, many, if not all, are unique to AbLV P120 and not shared by either Moloney MuLV Pr180gag-pol or Pr82gag.
Herpes Simplex Virus Glycoproteins: Participation of Individual Herpes Simplex Virus Type 1 Glycoprotein Antigens in Immunocytolysis and Their Correlation with Previously Identified Glycopolypeptides
Authors: Norrild B. , Shore S. L. , Nahmias A. J.
Published: January 2020

Tissue culture cells infected with herpes simplex type 1 virus express virus-specified glycoprotein antigens on the plasma membrane. Three of these have been previously identified and have been designated as Ag-11, Ag-8, and Ag-6. In the present study, immunoglobulins to each of the antigens were shown to be capable of mediating immunocytolysis in the presence of either complement (antibody-dependent complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cell-mediated cytotoxicity [ADCC]). Two herpes simplex virus type 1 strains, VR-3 and F, reacted similarly in the ADCC test in the presence of immunoglobulins to Ag-11, Ag-8, and Ag-6 in both infected Chang liver cells and HEp-2 cells. Anti-Ag-6, however, produced a lower ADCC reaction in HEp-2 cells than in Chang liver cells, suggesting differences in the Ag-6 surface expression in, or release from, these cells. Chang liver and HEp-2 cells infected with the MP mutant strain of herpes simplex virus type 1 showed reduced ADCC in the presence of anti-Ag-11 and anti-Ag-8, but no reactivity at all with anti-Ag-6. Crossed immunoelectrophoretic analysis showed that MP-infected cell extracts contain Ag-11 and Ag-8, but lack Ag-6. Polypeptide analysis of herpes simplex virus type 1 strains F, VR-3, and MP showed that Ag-11 consists of the glycoproteins gA and gB, that Ag-8 consists of gD, and that Ag-6 consists of gC. In conclusion, the present study demonstrates that either one of the glycoproteins (gC, gD, and a mixture of gA and gB) can function as a target for immunocytolysis and that the antibody preparation to gC (Ag-6) does not cross-react with any of the other glycoproteins.
Host transcription in bacteriophage P22-infected Salmonella typhimurium
Authors: Pipas J M , Reeves R H
Published: January 2020

The kinetics of host RNA synthesis, as measured by pulse-label kinetics and RNA-DNA hybridization, is inhibited 10-fold shortly after infection with bacteriophage P22. This early inhibition lasts through the first 6 min of infection and affects not only RNA synthesis but several other energy-requiring cellular functions. In lysogenic infections, the rate of bacterial transcription rapidly recovers to the value of uninfected controls. In lytic infections, the rate of host transcription increases only to about 20 to 25% of the original level, indicating a second mechanism for the inhibition of RNA synthesis in the lytic response. The early inhibition is multiplicity dependent, bhloramphenicol insensitive, and independent of phage gene 24. The lytic inhibition is dependent upon the expression of gene 24 but independent of gene 23.
Changes in three of the four coat proteins of oral polio vaccine strain derived from type 1 poliovirus
Authors: Kew O M , Pallansch M A , Omilianowski D R , Rueckert R R
Published: January 2020

Little is yet known about the nature, or extent, of the changes involved in attenuation of neurovirulent poliovirus. The tryptic comparison reported here, of coat proteins from the Sabin type 1 polio vaccine and parental Mahoney virus, provides a useful approach and affords some insight into this question. The main obstacle, separation of the labile proteins VP1 and VP2 in an intact state from the vaccine strain, was overcome by incorporating 3.5 M urea into an otherwise standard preparative gel electrophoresis system. Tryptic maps revealed six altered leucine-containing peaks: two in VP1, none in VP2, three in VP3, and one in VP4. It is estimated, after correcting for leucine-free peptides, that the coat protein sequences may have undergone some 10 to 13 amino acid replacements, roughly 1.5% of the total, in the course of attenuation leading to the vaccine strain.
ADP ribosylation of Escherichia coli RNA polymerase is nonessential for bacteriophage T4 development
Authors: Goff C G , Setzer J
Published: January 2020

The bacteriophage T4-induced alt and mod gene products covalently add ADP-ribose to the Escherichia coli RNA polymerase alpha polypeptides; phage carrying either an alt or a mod mutation are viable. A genetic cross between T4alt and T4mod phages yielded alt mod recombinant progeny which could not ADP ribosylate RNA polymerase at all, yet grew apparently normally. Thus, ADP ribosylation of RNA polymerase appeared to be nonessential for T4 development (at least in E. coli B/r and E. coli CR63), even though the phage has evolved two distinct enzymes to catalyze this reaction.
Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice
Authors: Ostertag W , Vehmeyer K , Fagg B , Pragnell I B , Paetz W , Le Bousse M C , Smadja-Joffe F , Klein B , Jasmin C , Eisen H
Published: January 2020

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.
Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c
Authors: Ellis R W , Hopkins N , Fleissner E
Published: January 2020

Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three of four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral DNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded GIX cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.
Comparison of Vesicular Stomatitis Virus Intracellular and Virion Ribonucleoproteins
Authors: Naeve Clayton W. , Kolakofsky Christiane M. , Summers Donald F.
Published: January 2020

Vesicular stomatitis virus ribonucleoproteins (RNP) obtained by a detergent treatment of purified virus (vRNP) or from infected HeLa cell cytoplasm (icRNP) were examined by sedimentation in sucrose or Renografin gradients in the presence or absence of EDTA. It was shown that vRNP and icRNP sediment at the same rate in sucrose and Renografin in the absence of EDTA; however, icRNP sedimented more slowly in the presence of EDTA than did vRNP. Polyacrylamide gel electrophoresis of the proteins of vRNA and icRNP recovered from EDTA-containing gradients demonstrated that both RNP structures contained L, N, and NS proteins in the same proportion. Electron microscopy of both RNP structures, in the absence of EDTA, demonstrated that both exist as helical structures ∼20 by 700 nm. However, in the presence of EDTA the icRNP was completely uncoiled with a mean length of 4,095 nm, whereas vRNP was hardly affected. The addition of excess Mg
2+
or Mn
2+
to uncoiled icRNP preparations partially restored the coiled configuration. These observations suggest that the change in sedimentation of icRNP in the presence of EDTA is due to a change from a coiled to an uncoiled conformation, that icRNP and vRNP are not structurally identical, and that icRNP must undergo a conformational change during maturation of VSV from the 20-by-700-nm intracellular form to the 50-by-175-nm form found in intact virus. The icRNP containing L, N, and NS proteins (icRNP
L,N,NS
) and icRNP containing only N protein (icRNP
N
), prepared by centrifugation of icRNP
L,N,NS
in CsCl to remove L and NS, were compared by cosedimentation in sucrose gradients. There was a decrease in sedimentation rate of icRNP
N
due to loss of L and NS. This sedimentation difference was also apparent in the presence of EDTA; however, both icRNP
L,N,NS
and icRNP
N
sedimented at a much slower rate in the presence of EDTA, and by electron microscopy both were completely uncoiled. These observations suggest that N protein alone is responsible for the 20-by-700-nm coiled structure and that the divalent cation interactions disrupted by EDTA are N-N or N-RNA interactions. These results are discussed with regard to vesicular stomatitis virus maturation.
Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen
Authors: Soule Howard R. , Lanford Robert E. , Butel Janet S.
Published: January 2020

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoαPM, MoαNuc), glutaraldehydefixed SV40-transformed cells (HaαH-50-G, MoαVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaαT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaαT, MoαT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaαS, MoαS). Immunological reagents prepared against cell surface (MoαPM, HaαS, MoαS, HaαH-50-G, MoαVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoαNuc) or whole-cell determinants (HaαT, MoαT, HAF, RaαT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaαS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaαS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoαDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaαT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.
Integrated Moloney murine leukemia virus DNA studied by using complementary DNA which does not recognize endogenous related sequences
Authors: Bacheler L T , Fan H
Published: January 2020

By preannealing a radioactive, representative Moloney murine leukemia virus (M-MuLV) cDNA with large excesses of AKR 70S viral RNA, an M-MuLV-specific cDNA has been prepared. When hybridized to restriction enzyme fragments of M-MuLV-infected mouse cell DNA, the preannealed probe recognizes integrated M-MuLV DNA and does not recognize endogenous related DNA sequences found in uninfected mouse cells. The viral DNA sequences recognized by the preannealed probe are spread throughout the viral genome, although some sequences are recognized less efficiently. By using this preannealed probe, multiple integrations of M-MuLV DNA have been detected in infected fibroblasts and in an M-MuLV-induced tumor. Integrated viral DNA fragments smaller than the complete viral genome have also been detected. By using this preannealed probe to examine a mass-infected culture of mouse fibroblasts, no evidence for a strongly preferred site for M-MuLV integration could be found.
A clear-plaque mutation of bacteriophage M13 affects the regulation of viral DNA synthesis
Authors: Farber M B , Ray D S
Published: January 2020

A clear-plaque mutation (c2) of bacteriophage M13 has been shown to affect the regulation of viral DNA synthesis. This mutation increases the amount of the duplex replicative form DNA per cell while decreasing the synthesis of viral single strands. The relative synthesis of the M13 gene 5 protein is approximately half that observed in wild-type infections, suggesting that the effect of the c2 mutation on the regulation of viral DNA synthesis is a result of reduced expression of gene 5.
Late modifications of simian virus 40 chromatin during the lytic cycle occur in an immature form of virion
Authors: La Bella F , Vesco C
Published: January 2020

Two main modifications of the simian virus 40 chromatin were found to occur during the lytic cycle. One was the progressive increase in the acetylation level in the four non-H1 histones as the 75S deoxynucleoprotein complexes (minichromosomes) became assembled into heavier structures. The other was the final elimination from viral chromatin of histone H1. An important stage in the course of these changes was represented by an intracellular simian virus 40 particle, in which the virus-coded proteins were already assembled, but properties distinct from those of mature virions were still present. This particle resembled the mature virions in morphology, sedimentation rate, and buoyant density. It was distinguished by the instability, the presence of histone H1, the uptake of radioactive acetate, and the lower infectivity. Its significance appears to be that of an immature virion on the basis of these characters and of the consistent kinetic behavior during the lytic cycle.
Ultrastructural characterization of an early, nonstructural polypeptide of herpes simplex virus type 1
Authors: Cabral G A , Courtney R J , Schaffer P A , Marciano-Cabral F
Published: January 2020

An immunoperoxidase procedure was employed to study the expression of a large-molecular-weight, virus-induced polypeptide (VP175; molecular weight, 175,000) at the light and electron microscopic levels in Vero cells infected with herpes simplex virus type 1 or with tsB2, a DNA-negative, temperature-sensitive mutant of herpes simplex virus type 1. In cells infected with herpes simplex virus type 1 and in cells infected with tsB2 at the permissive temperature (34 degrees C), VP175 was found within the nucleus. The protein was detected as early as 2 h postinfection and, by 3 h postinfection, was generally distributed in a marginated pattern contiguous with, and extending from, the inner lamella of the nuclear membrane. At 6 h postinfection, protein accumulations were dispersed throughout the nucleus, and, by 9 h postinfection, these accumulations tended to be localized in a marginated pattern near the nuclear membrane. It was also noted that, at 9 h postinfection, under permissive conditions, VP175 was not found in association with nucleocapsids or enveloped particles. In contrast, in cells infected with tsB2 at the nonpermissive temperature (39 degrees C) and harvested at 6 or 9 h postinfection, accumulations of VP175 were identified not only within the nucleus, but also within the cytoplasm in the form of annular or globular aggregates. These aggregates consisted of a granular matrix and were not bound by membranes.
Short-lived minus-strand polymerase for Semliki Forest virus
Authors: Sawicki D L , Sawicki S G
Published: January 2020

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.
Post-Translational Proteolytic Cleavage of In Vitro-Synthesized Turnip Yellow Mosaic Virus RNA-Coded High-Molecular-Weight Proteins
Authors: Morch Marie-Dominique , Benicourt Claude
Published: January 2020

In a reticulocyte lysate, turnip yellow mosaic virus genomic RNA directs the synthesis of two proteins with molecular weights of 150,000 (150K) and 195K. We present evidence that the larger protein is processed in vitro, after its completion, in at least three fragments. The NH
2
-terminal fragment (82K) and the COOH-terminal fragment (78K) have been well characterized by different methods. The fact that the 150K protein is not cleaved in vitro, although it contains the regions that are processed in the 195K protein, could be of fundamental biological significance for the expression of the viral genes: a single polypeptide chain could be processed in several ways, leading to different peptides with distinct biological activities.
Baseplate protein of bacteriophage T4 with both structural and lytic functions
Authors: Kao S H , McClain W H
Published: January 2020

Analyses of a new bacteriophage T4 mutant that permits lysis of infected cells in the absence of e lysozyme showed that the strain carried a suppressor mutation in gene 5, a gene whose polypeptide product (gp5) is an integral component of the virion baseplate. Indirect experiments indicated that cell lysis was caused by the lytic action of mutant gp5. With regard to the physiological role of normal gp5, we speculate that it functions in the initiation of infection by catalyzing local cell wall digestion to facilitate penetration of the tail tube through the cell envelope. The proposed lytic activity of gp5 may also be responsible for the well-known phenomenon of lysis from without observed with T4.
Characterization of avian myeloblastosis-associated virus DNA intermediates
Authors: Bergmann D G , Souza L M , Baluda M A
Published: January 2020

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.
Relationship of retroviruses isolated from human leukemia tissues to the woolly monkey-gibbon ape leukemia viruses
Authors: Sahagan B G , Haseltine W A
Published: January 2020

Retroviruses have been isolated from the tissues of human leukemia patients. Previous studies have shown that these isolates share some antigenic determinants with the family of viruses isolated from the woolly monkey and gibbon ape and that they exhibit partial nuclei acid homology with this same group of viruses. We have compared the RNAs of the viruses by two-dimensional polyacrylamide gel electrophoresis of the large RNase T1-resistant oligonucleotides. The degree of sequence identity between the RNAs was determined by the similarity of their RNase T1-resistant oligonucleotide pattern on gels, fingerprints, and in some cases by partial sequence analysis of individual oligonucleotides. This technique permits us to determine the degree of sequence identity among related RNA species. From our studies we conclude that viruses isolated from the tissues of two human leukemia patients, A1476 and SKA 21-3, as well as some subcultures of a virus isolated from the leukemic tissues of a third patient, HL23V, are closely related to the wooly monkey virus. However, the fingerprints of other HL23 viral isolates are very similar to that of GaLVSF, a gibbon ape leukemia virus isolated from a lymphosarcoma.
Heterogeneity of the genome-linked protein of foot-and-mouth disease virus
Authors: King A M , Sangar D V , Harris T J , Brown F
Published: January 2020

The genome-linked protein of foot-and-mouth disease virus was examined by electrofocusing in polyacrylamide gels. Two proteins of different charge and amino acid composition were found. The tryptic peptide maps of the proteins were dissimilar. The possible relationship between the two proteins is discussed.
Adenovirus regulation of simian virus 40 macromolecular synthesis
Authors: Goldman N , Khoury G
Published: January 2020

At low multiplicities of infection (1 to 5 PFU/cell), human adenoviruses grew inefficiently in monkey kidney cells as the result of a late transcriptional and/or translational defect. Through an unknown mechanism, coinfection with simian virus 40 (SV40) could overcome this block. Coincident with the onset of adenovirus DNA replication in coinfected monkey cells, however, there was a marked inhibition of SV40 DNA replication. Since this inhibition in SV40 DNA synthesis occurred in the presence of normal SV40 transcription and protein synthesis, we suggest that adenovirus competes with SV40 for molecules specifically required for DNA synthesis (e.g., SV40 T-antigen). At high multiplicities of adenovirus infection (50 PFU/cell), adenoviruses could grow efficiently in monkey kidney cells. In cells coinfected under these conditions, SV40 transcription was generally inhibited, as shown by a decrease in nuclear and cytoplasmic SV40 mRNA.
Late-passage SC-1 cells resistant to N- and B-tropic viruses but sensitive to NB-tropic viruses
Authors: Pincus T
Published: January 2020

A late-passage derivative of SC-1 cells with stable relative resistance to N- and B-tropic viruses but with retained sensitivity to NB-tropic viruses has been identified. Dual resistance was seen with the XC plaque assay, as well as reverse transcriptase assay and radioimmunoassay for viral p30. Evidence was found for resistance in clonal derivatives. These findings suggest a possible instability in determinants of cellular resistance to endogenous retroviruses and may be useful in further analysis of resistance to retrovirus infection.
Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA
Authors: Bailey J N , Dembinski D R , McAllister W T
Published: January 2020

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.
Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus
Authors: Ito Y , Spurr N , Griffin B E
Published: January 2020

A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.
Macromolecular requirements for abrogation of Fv-1 restriction by murine leukemia viruses
Authors: Bassin R H , Gerwin B I , Levin J G , Duran-Troise G , Benjers B M , Rein A
Published: January 2020

The molecular basis of abrogation of Fv-1 restriction in mouse cells by murine leukemia virus was investigated. Two different lines of experimentation indicated that high-molecular-weight viral RNA is required for abrogation. First, the decay of abrogating ability of virus stocks heated at 43 degrees C was quantitatively correlated with a loss of intact virion 35S RNA. Second, Act D virions, which lack such RNA although they contain normal structural proteins, failed to abrogate. These findings imply that abrogation does not result from the mere entry of virion structural proteins into a cell. Additional data indicate that the role of viral RNA in abrogation is not that of a template for DNA synthesis. Virus particles lacking reverse transcriptase activity as a result of either mutation or heat inactivation exhibit abrogating activity even though they do not synthesize detectable viral DNA. In addition, abrogation was shown to take place in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. Thus, abrogation does not depend on viral or cellular DNA synthesis, and the role of viral RNA in this process must involve some other function. The nature of this viral function and its occurrence in Fv-1 permissive cells are discussed.
Structure and processing of the mouse mammary tumor virus glycoprotein precursor pr73env
Authors: Dickson C , Atterwill M
Published: January 2020

The polyprotein precursor to the envelope glycoproteins of mouse mammary tumor virus was investigated by using subcellular fractionation procedures, pactomycin mapping techniques, tunicamycin inhibition of glycosylation, and endo-beta-N-acetyl glucosaminidase H-catalyzed removal of glycosylated residues in order to characterize the biosynthesis and processing of the precursor. The results suggest that the precursor (Pr73env) is synthesized on the rough endoplasmic reticulum as a transmembrane protein, with the carboxyl terminus remaining on the cytoplasmic side. The apoprotein as an estimated molecular weight of 60,000 and acquires five core oligosaccharide units during synthesis. Cleavage of the precursor precedes the secondary glycosylation steps and therefore probably occurs before transport to the plasma membrane. However, a minor population of Pr73env containing complex oligosaccharides was also found in the plasma membrane. The order of the glycoproteins in the precursor, as determined by pactomycin mapping, in NH2-gp52-gp36-COOH.