bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylation and amplification of mouse mammary tumor virus DNA in normal, premalignant, and malignant cells of GR/A mice
Authors: Fanning T G , Vassos A B , Cardiff R D
Published: January 2020

The methylation and amplification of mouse mammary tumor virus (MuMTV) proviral DNA was investigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylation pattern was examined with the restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heart was highly methylated. Proviral DNA was somewhat undermethylated in mammary gland cells from virgin and lactating mice and extensively undermethylated in cells from premalignant outgrowths, pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme SacI was used to detect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammary gland cells from virgin mice. Some mammary gland cells from lactating mice appeared to contain additional copies of the endogenous, highly oncogenic GT-MTV-2 provirus. Premalignant outgrowth, pregnancy-dependent tumor, and pregnancy-independent tumor cells contained an average of two to three additional copies per cell of the GT-MTV-2 provirus. Thus, neoplasia in GR/A mice was directly associated with quantized increases in MuMTV proviral DNA undermethylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme analysis suggested that premalignant outgrowths and pregnancy-dependent tumors both consisted largely of heterogenous cell populations, although some evidence of clonal dominance was detected.
Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure
Authors: Ben-Asher E , Bratosin S , Aloni Y
Published: January 2020

Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.
Small t protein of simian virus 40 is required for dense focus formation in a rat cell line
Authors: Sugano S , Yamaguchi N , Shimojo H
Published: January 2020

Incomplete foci were formed in a Fischer rat cell line infected with simian virus 40 detection mutants encoding defective small t antigen. The foci were small and rarely piled up. The fraction of DNA-synthesizing cells in the foci was about half of that induced by wild-type virus.
Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1
Authors: Hewlett M J , Axelrod J H , Antinoro N , Feld R
Published: January 2020

We isolated six temperature-sensitive mutants of poliovirus type 1 (Mahoney) by hydroxylamine mutagenesis and replica plating at 31, 33 (permissive), and 39 degrees C (restrictive). One of these mutants, designated tsB9, was chosen for more detailed examination. tsB9 accumulated 25% of the wild-type amount of virus-specific RNA at the restrictive temperature. We found that tsB9 was not able to synthesize mature, 35S single-stranded RNA at the restrictive temperature. In spite of the absence of significant RNA synthesis, tsB9 retained the ability to inhibit host protein synthesis during infection at 39 degrees C at about the same rate as wild-type virus.
mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay
Authors: Kroath H , Shatkin A J
Published: January 2020

Reovirus mRNA 5'-terminal caps were 3'-radiolabeled with pCp and as affinity probes for proteins with cap binding activity. A rapid, simple, and sensitive blot assay was devised that could detect cellular cap binding protein in a complex polypeptide mixture. By using this method, cap binding activity was found in detergent-treated influenza virus but not in reovirus or vaccinia virus. Preincubation of capped reovirus mRNA with purified cellular cap binding protein reduced its primer effect on influenza transcriptase, whereas priming by ApG was not affected. The results indicate that influenza transcriptase complexes include cap-recognizing proteins that are involved in the formation of chimeric mRNAs.
Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus
Authors: Birkenmeier E H , Bonner T I , Reynolds K , Searfoss G H , Todaro G J
Published: January 2020

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.
Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei
Authors: Tapper Douglas P. , Anderson Stephen , DePamphilis Melvin L.
Published: January 2020

The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI
*
) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished three- to fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80% completed SV40(RI) DNA. Failure to convert SV40(RI
*
) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with their nascent DNA strands interrupted in the termination region [SV40(II
*
) DNA] accumulated with unjoined Okazaki fragments. Thus, separation of sibling chromosomes still occurred, but gaps remained in the terminal portions of their daughter DNA strands. These and other data support a central role for SV40(RI
*
) and SV40(II
*
) DNAs in the completion of viral DNA replication.
DNA of herpesvirus pan, a third member of the Epstein-Barr virus-Herpesvirus papio group
Authors: Heller M , Gerber P , Kieff E
Published: January 2020

The DNA of herpesvirus pan, a primate B-lymphotropic herpesvirus, shares about 40% well-conserved sequence relatedness with Epstein-Barr virus (EBV) and herpesvirus papio DNAs. Labeled cloned fragments from the EBV recombinant DNA library were cross hybridized to blots of EcoRI, XbaI, and BamHI restriction endonuclease fragments of herpesvirus pan DNA to identify and map homologous sequences in the herpesvirus pan genome. Regions of colinear homology were demonstrated between 6 x 10(6) daltons and 108 x 10(6) daltons in the DNAs. The structural organization of herpesvirus pan DNA was similar to the format of Epstein-Barr virus and herpesvirus papio DNAs. The DNA consists of two domains of largely unique sequence complexity, a segment US of 9 x 10(6) daltons and a segment UL of 88 x 10(6) daltons. US and UL are separated by a variable number of tandem repetitions of a sequence IR (2 x 10(6) daltons). There was homology between DNA which mapped at 26 to 28 x 10(6) daltons and 93 to 95 x 10(6) daltons in UL. The terminal reiteration component, TR, of herpesvirus pan DNA and sequences which mapped to the left of 6 x 10(6) daltons and to the right of 108 x 10(6) daltons had no detectable homology with the corresponding regions of Epstein-Barr virus DNA.
DNA polymerase requirements for parvovirus H-1 DNA replication in vitro
Authors: Kollek R , Tseng B Y , Goulian M
Published: January 2020

An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.
Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins
Authors: Briedis D J , Lamb R A
Published: January 2020

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.
Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells
Authors: Hansen J , Etchison D , Hershey J W , Ehrenfeld E
Published: January 2020

Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.
Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus
Authors: Shih T Y , Weeks M O , Gruss P , Dhar R , Oroszlan S , Scolnick E M
Published: January 2020

The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.
Synthesis of murine leukemia viral DNA in vitro: evidence for plus-strand DNA synthesis at both ends of the genome
Authors: DesGroseillers L , Rassart E , Zollinger M , Jolicoeur P
Published: January 2020

We studied the synthesis of B-tropic murine leukemia viral DNA in vitro by detergent-disrupted virions. The reaction products (detected by the Southern transfer technique) included full-length, infectious, double-stranded DNA and several subgenomic fragments. Restriction endonuclease analysis and hybridization and specific probes revealed two classes of subgenomic fragments: some were derived from the right end of the genome, and some were derived from the left end. Most of the fragments harbored one long terminal repeat copy at their ends, suggesting that they were initiated correctly. S1 nuclease and restriction endonuclease treatments of these fragments indicated that a single-stranded gap was present near the first initiation site of plus strong-stop DNA. The treatments also suggested the presence of a second initiation site flanked by a single-stranded gap 0.9 kilobase pairs from the right end of the genome. Our data clearly show that plus-strand DNA is synthesized at both ends of the genome, by using plus strong stop as the first initiation site and additional initiation sites.
Glycoproteins of friend murine leukemia virus: separation and NH2-terminal amino acid sequences of gp69 and gp71
Authors: Linder D , Stirm S , Schneider J , Hunsmann G , Smythers G , Oroszlan S
Published: January 2020

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.
Nucleotide sequences at the phi X gene A protein cleavage site in replicative form I DNAs of bacteriophages U3, G14, and alpha 3
Authors: Heidekamp F , Baas P D , Jansz H S
Published: January 2020

Gene A protein, a bacteriophage phi X174-encoded endonuclease involved in phi X replicative form (RF) DNA replication, nicks not only phi X RFI DNA but also RFI DNAs of several other spherical single-stranded DNA bacteriophages. The position of the phi X gene A protein nick and the nucleotide sequence surrounding this site in RF DNAs of the bacteriophages U3, G14, and alpha 3 were determined. Comparison of the nucleotide sequences which surround the nick site of the gene A protein in RF DNAs of phi X174, G4, St-1, U3, G14, and alpha 3 revealed that a strongly conserved 30-nucleotide stretch occurred in RF DNAs of all six phages. However, perfect DNA sequence homology around this site was only 10 nucleotides, the decamer sequence CAACTTGATA. The present results support the hypothesis that, for nicking of double-stranded supercoiled DNA by the phi X gene A protein, the presence of the recognition sequence CAACTTGATA and a specific gene A protein binding sequence upstream from the recognition sequence are required. The sequence data obtained so far from phages U3, G14, St-1, and alpha 3 have been compared with the nucleotide sequences and amino acid sequences of both phi X and G4. According to this comparison, the evolutionary relationship between phages G4, U3, and G14 is very close, which also holds for phages alpha 3 and St-1. However, the two groups are only distantly related, both to each other and to phi X.
Epstein-Barr Virus-Lymphoid Cell Interactions III. Effect of Concanavalin A and Saccharides on Epstein-Barr Virus Penetration
Authors: Khélifa Ridha , Menezes José
Published: January 2020

To study some aspects of Epstein-Barr virus (EBV) penetration into target cells, the effect of concanavalin A (ConA) and various saccharides on virus infectivity and cell susceptibility to EBV infection was examined. ConA treatment of the target cells, EBV, or EBV-cell complexes was found to inhibit virus antigen expression. Several control experiments with α-
d
-methyl-mannoside elution of ConA, removal of nonfused EBV particles from the cell surface by trypsin treatment, and addition of ConA at different times postinfection were performed to define the site of ConA action on EBV infection. ConA appeared to have a dual action: (i) it inhibited EBV binding to virus receptors, and (ii) it blocked the penetration of receptor-bound virus into target cells at a trypsin-sensitive stage, thus indicating that ConA prevented the fusion of viral envelope with the target cell membrane. A high sucrose concentration (0.25 M), known to inhibit cell membrane movements, was also found to block EBV penetration at a trypsinsensitive stage, thus suggesting the implication of cell membrane movements and underlying activities (or both) in viral envelope fusion. Lower concentrations of various monosaccharides (0.12 M) did not influence EBV infection. Under conditions of ConA treatment that did not influence EBV infectivity and target cells susceptibility, ConA was able to mediate virus binding to EBV receptornegative cell lines, but no virus antigens were expressed in these cells. These observations reinforced the idea that the mere attachment of EBV to lymphoid cells is not sufficient to lead to infection. In light of the present and previously published data, we postulate the existence of a specific cellular mechanism that allows the penetration of EBV into the target (B) lymphocyte.
Properties of a novel thymidine kinase induced by an acyclovir-resistant herpes simplex virus type 1 mutant
Authors: Larder B A , Darby G
Published: January 2020

The acyclovir-resistant mutant of herpes simplex virus type 1, SC16 S1, induced reduced levels of thymidine kinase activity (ca. 25% reduction) in infected cells. The activity appeared with kinetics similar to that in wild type-infected cells, and pulse-labeling experiments showed that the thymidine kinase polypeptide was synthesized at a similar rate. We showed that the enzyme was virus specific by inactivating it with antiserum raised against herpes simplex virus-infected cell proteins. The enzyme induced by the mutant had reduced electrophoretic mobility in nondenaturing gels, decreased thermal stability, and decreased affinity for several different substrates (assessed by measurement of Km values) compared with the enzyme induced by the wild type. From the data obtained we conclude that the thymidine kinase induced by the mutant has an altered specificity, probably resulting from an amino acid substitution which affects the primary binding site for nucleosides and nucleoside analogs.
Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus
Authors: Smith G W , Hightower L E
Published: January 2020

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.
Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells
Authors: Anderson K P , Klessig D F
Published: January 2020

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.
Genetic analysis of murine hepatitis virus strain JHM
Authors: Leibowitz J L , DeVries J R , Haspel M V
Published: January 2020

We performed a genetic analysis of 37 temperature-sensitive mutants of murine hepatitis virus strain JHM. Of our mutants, 32 did not induce murine hepatitis virus-specific RNA synthesis in infected cells at the restrictive temperature, 39 degrees C. By complementation testing we have identified at least seven nonoverlapping complementation groups. Six of the genes identified in this way are required for murine hepatitis virus-specific RNA synthesis. The seventh complementation group is made up of five mutants which induced virus-specific RNA synthesis at 39 degrees C.
Evidence for simian virus 40 late transcriptional control: mixed infections of wild-type simian virus 40 and a late leader deletion mutant exhibit trans effects on late viral RNA synthesis
Authors: Alwine J C
Published: January 2020

Mixed infections involving equal multiplicities of wild-type simian virus 40 and viable deletion mutant dl861 resulted in decreased cytoplasmic levels of wild-type-derived male mRNA, as well as very low to undetectable levels of mutant-derived late mRNA, as compared with individual infections. The dl861 deletion removes 16 to 25 base pairs from the late leader region. This deletion was shown to be the direct cause of the mixed-infection effect; replacement of the deletion with wild-type sequences restored normal levels of late mRNAs in mixed infections. Other viral functions, e.g., early gene expression and replication, were found to be unaffected by the dl861 deletion. Further examination of the mixed-infection effect showed that the levels of unspliced nuclear precursors of late mRNA, derived from both the mutant and wild-type genomes, were decreased or undetectable, in accord with the cytoplasmic results. Thus, the effect appears to be occurring at the transcriptional level. These data demonstrate a trans-acting effect on late transcription, which is detectable due to the presence of the dl861 mutant in the mixed infection. This finding is indicative of a diffusible factor which exerts a control on simian virus 40 late gene expression at the transcriptional level. A model for positive control of simian virus 40 late gene expression is presented.
ML antigen of DBA/2 mouse leukemias: expression of an endogenous murine mammary tumor virus
Authors: Racevskis J , Sarkar N H
Published: January 2020

Spontaneous, transplantable leukemias of DBA/2 mice express an antigen (ML) which cross-reacts with antigens of murine mammary tumor virus (MuMTV). The MuMTV cross-reactive antigen of the DBA/2 leukemias (ML cells) was found to be a glycoprotein of 78,000 molecular weight containing antigenic determinants of the major MuMTV glycoprotein gp52. No MuMTV particles were produced by the ML cells, although they did contain type A particles--the pronucleocapsids of MuMTV. The ML antigen appeared to be an aberrant form of the intracellular MuMTV env precursor molecular prgp70, which was not processed properly but instead acquired extra carbohydrate groups and was expressed in uncleaved form on the cell surface. Isolation of MuMTV core protein p28 from the leukemic cells and subsequent tryptic peptide mapping analysis showed that the p28 from leukemia cells differed from the p28 of MuMTV isolated from DBA/2 mouse milk. These observations indicate that the MuMTV expressed in DBA/2 leukemic spleen cells is of a different strain than the virus secreted in lactating mammary glands of DBA/2 mice and probably represents the expression of an endogenous DBA/2 provirus.
Antigenic and genetic characterization of a novel hemagglutinin subtype of influenza A viruses from gulls
Authors: Hinshaw V S , Air G M , Gibbs A J , Graves L , Prescott B , Karunakaran D
Published: January 2020

Influenza A virus isolates from ring-billed, Franklin, blackback, and herring gulls in the United States possess a hemagglutinin (HA) distinct from the 12 reference HA subtypes. Serological assays (hemagglutination inhibition and double-immunodiffusion) with specific antisera to reference strains and to a representative gull isolate showed that the HA of the gull virus was not antigenically related to that of any known subtype. The gull virus did not replicate in ducks or chickens but did replicate in ferrets. Comparison of the nucleotide sequences (and deduced amino acid sequences) of the 3' 20% of the HA genes of these viruses indicates that the gull viruses represent a genetically distinct group. We propose that this HA, which has been detected only in gull isolates thus far, be called the H13 subtype.
Mutant identifying a third recombination group in a bunyavirus
Authors: Pringle C R , Iroegbu C U
Published: January 2020

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.
Change of DNA near the origin of replication enhances the transforming capacity of human papovavirus BK
Authors: Watanabe S , Yoshiike K
Published: January 2020

A turbid-plaque-forming mutant (pm522) of human papovavirus BK, which has a small deletion at about 0.7 map unit and grows somewhat more slowly in human cells than does wild-type BK virus, transformed hamster and rat cells in culture much more efficiently than did wild-type virus. Another plaque morphology mutant, pm525, forming turbid plaques larger than those of pm522 also had a high transforming capacity. The similar difference in transforming capability between wild-type and plaque morphology viruses was observed with DNAs extracted from virions. Recombinant viruses were constructed from the wild-type DNA fragment lacking HindIII-C (0.62 to 0.73 map unit) and pm522 HindIII-C (including the origin of replication) by the molecular cloning method. Characterization of the recombinants showed that the change near the origin of DNA replication was responsible both for the altered plaque morphology and for the enhanced transforming capacity of the BK virus mutant.
Black Beetle Virus: Messenger for Protein B Is a Subgenomic Viral RNA
Authors: Friesen Paul D. , Rueckert Roland R.
Published: January 2020

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein α (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected
Drosophila
cells. Two of these proteins, A and α, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and α cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.
RNA synthesis by Newcastle disease virus temperature-sensitive mutants in two RNA-negative complementation groups
Authors: Peeples M E , Rasenas L L , Bratt M A
Published: January 2020

The temperature-sensitive RNA-negative mutants of Newcastle disease virus comprise two complementation groups, group A (seven members) and group E (one member). The RNA-synthesizing activities of four representative members of group A and the single member of group E were compared with the activity of the wild type. These mutants were defective to varying extents in primary transcription at the nonpermissive temperature, ranging from mutant A1, which had no activity, to mutant E1, which lost only 50% of its activity. All of the mutants were also defective in a postprimary transcriptive process since after preincubation at the permissive temperature in the presence of cycloheximide, there was no subsequent RNA synthesis at the nonpermissive temperature upon removal of the cycloheximide. Similarly, in experiments in which cycloheximide was not used, shifts from the permissive temperature to the nonpermissive temperature before 3 h postinfection did not result in RNA synthesis. However, later shifts to the nonpermissive temperature did allow RNA synthesis. With the exception of mutant A1, all of the mutants maintained this RNA-synthetic ability for at least 3 h, suggesting that RNA synthesis from progeny genomes was not the major postprimary transcriptive defect in these mutants. In contrast, the RNA-synthetic ability of mutant A1 rapidly decayed at the nonpermissive temperature, suggesting that the A gene product is involved in RNA synthesis from progeny genomes. The postprimary transcriptive defect(s) of the other mutants may be in the processing or stability of a protein, in the processing of mRNA, or in replication. Plaque-forming revertants (ts+) of all of the mutants coreverted for RNA synthesis. This finding strengthens the relationship between temperature sensitivity for plaquing and both the primary and postprimary RNA-negative phenotypes.
Site-specific phosphorylation regulates the transcriptive activity of vesicular stomatitis virus NS protein
Authors: Hsu C H , Morgan E M , Kingsbury D W
Published: January 2020

In vitro transcription by vesicular stomatitis virus nucleocapsids is inhibited by enzymatic dephosphorylation of the NS protein. We provide evidence that specific, partial dephosphorylation of NS molecules is the only detectable change in nucleocapsids treated with bacterial alkaline phosphatase under conditions that prevent the action of adventitious protease. Dephosphorylation appeared to affect only the rate of transcription; there were no changes in sedimentation rates of transcripts. To identify the sites of phosphorylation required for NS activity in transcription, we examined phosphopeptides produced by chymotrypsin digestion of the two electrophoretic classes of NS molecules found in virions and infected cells. The electrophoretically slower class, NS1, abundant in the intracellular soluble pool, has a lower activity in transcription; it contained six chymotryptic phosphopeptides. Five of these peptides contained both phosphoserine and phosphothreonine, indicating that this peptide cluster represents at least 11 separate sites of phosphorylation. In the electrophoretically faster nucleocapsid-associated NS2 class of molecules, which support a higher rate of transcription, another group of eight phosphopeptides was superimposed on this pattern. Two of these peptides contained both phosphoserine and phosphothreonine, so this cluster of peptides represents at least 10 additional phosphorylation sites. These sites were especially sensitive to dephosphorylation by bacterial alkaline phosphatase. One or more of them appears to be responsible for the higher transcription rates medicated by NS2 molecules.
Herpesvirus saimiri strain variability
Authors: Desrosiers R C , Falk L A
Published: January 2020

Herpesvirus saimiri was isolated from 22 squirrel monkeys by cocultivation of peripheral lymphocytes with permissive owl monkey kidney monolayer cells. Comparison of virion DNA fragments produced from restriction endonuclease digestion was used as a sensitive measure of strain variability. Although all isolates contained similarities and common features, 19 of the 22 were readily distinguished. Three of the isolates, however, were indistinguishable and possibly were related epidemiologically. Distinct subtypes of H. saimiri were not evident by these criteria; Peruvian, Colombian, Guyanan, and Bolivian squirrel monkeys yielded isolates without characteristic features peculiar to the geographic region. Three of three colony-born squirrel monkeys that were tested yielded a strain of virus distinct from that obtained from the mother. In separate experiments, two of three animals chosen at random yielded a strain of virus different from that originally obtained 16 and 22 months previously; only one of the three animals examined yielded the same strain of virus 22 months after the original isolation. The degree of restriction endonuclease fragment variability among H. saimiri strains appeared to be greater than previously observed for other herpesviruses.
Specifically unmethylated cytidylic-guanylate sites in Herpesvirus saimiri DNA in tumor cells
Authors: Desrosiers R C
Published: January 2020

The restriction endonucleases MspI (CCGG), HpaII (CCGG), FnuDII (CGCG), and HaeIII (GGCC) were used to study the methylation of Herpesvirus saimiri DNA in tumor cells taken directly from tumor-bearing animals. No evidence was found for methylation of the 5' terminal C in the sequence CCGG or of the internal C in the sequence GGCC, but extensive methylation of CG was detected. Fifteen HpaII sites and 17 FnuDII sites were detected in the unique DNA region of the H. saimiri strain used. Twenty-eight of the 32 sites were methylated in greater than 90% of the viral DNA molecules in tumor cells, but the remaining 4 sites were unmethylated in greater than 95% of the viral DNA molecules in tumor cells. The locations of the four specifically unmethylated sites were mapped and appeared to be identical in the four different induced leukemias examined (one owl monkey and three white-lipped marmosets). The nonproducer 1670 tumor cell line, in continuous passage for over 7 years, contained four similar specifically unmethylated sites. Possibilities for the physiological significance of the unmethylated sites are discussed.
Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis
Authors: Kremsdorf D , Jablonska S , Favre M , Orth G
Published: January 2020

The DNAs of the human papillomaviruses (HPVs) associated with the benign lesions of two patients suffering from epidermodysplasia verruciformis (patients JD and JK) were analyzed by using 12 restriction endonucleases. None of the restriction endonucleases were one-cut enzymes for the HPV DNA obtained from patient JD, referred to as the prototypical HPV-5, whereas five of them were one-cut enzymes for the DNA of the major virus found in patient JK, referred to as HPV-9. The molecular cloning of the two fragments resulting from the cleavage of HPV-5 DNA by endonuclease HindIII and of the single fragment obtained after treatment of HPV-9 DNA with endonuclease BamHI was performed in Escherichia coli after the fragments were inserted in plasmid pBR322. A cleavage map of the two cloned genomes was constructed. Little sequence homology (4 to 5%) was detected between HPV-5 and HPV-9 DNAs by DNA-DNA hybridization experiments in liquid phase at saturation; this homology was reproducibly higher than that (2 to 3%) detected under the same conditions between these DNAs and HPV-1a DNA. In addition, blot hybridization experiments performed under stringent conditions showed no or little sequence homology between the DNAs of HPV-5 and HPV-9 and those of HPV prototypes of types 1, 2, 3, 4, and 7 associated with skin warts. These results confirm that HPV-5 and HPV-9 are two distinct HPV types.
Charge microheterogeneity of the major capsid protein of polyoma virus
Authors: Hare J D , King V L
Published: January 2020

The behavior in isoelectric focusing of the major capsid polypeptide VPI of several strains of polyoma virus was studied. Two previously recognized phenomena were reexamined, namely, (i) the separation of the VP1 polypeptide into multiple subspecies differing only slightly from each other in apparent isoelectric point and (ii) strain differences in the overall apparent net charge of the family of VP1 subspecies. It was found that the pattern of subspecies was reproducible when focusing was initiated from either the basic or acidic region of the gel, keeping the ampholyte mixture constant. However, individual subspecies were unstable, and labeled polypeptide could be shifted dramatically by either refocusing of separated subspecies or by altering the concentration of ampholytes. These findings suggest that protein-protein and protein-ampholyte interactions play an important role in the generation of this charge heterogeneity. The basis for the overall charge difference between the VP1 of 3049 virus and several other strains (lpD, lpS, ts59, and A2) was studied, using recombinant viruses constructed of specific sequences derived from 3049 and lpD genomes. The portion of the VP1 polypeptide carrying the altered charge could be mapped to the body of the molecule 3' to the HindIII site at 45.0 map units (3,918 base pairs). This clearly segregates the VP1 charge phenotype from the cyc phenotype of 3049 in which capsid proteins are overproduced and accumulate in the cytoplasm of infected cells.
Avian Reovirus Polypeptides: Analysis of Intracellular Virus-Specified Products, Virions, Top Component, and Cores
Authors: Schnitzer Thomas J. , Ramos Teresa , Gouvea Vera
Published: January 2020

Avian reovirus-specified polypeptides can be separated into three size classes: large (λ), medium (μ), and small (σ), similar to those of the mammalian reoviruses. A nomenclature has been proposed to indicate the individual polypeptides within each size class by progressive alphabetical subscripts. Three λ polypeptides (λ
A
, λ
B
, and λ
C
) are found in infectious viral particles and have molecular weights of 145,000, 130,000, and 115,000, respectively. All are present in core preparations, and two (λ
A
and λ
B
) appear to be exposed at the surface of the virion. Two μ polypeptides can be distinguished in purified virus (μ
A
, 72,000 daltons; μ
B
, 70,000 daltons), and another is occasionally evident by immunoprecipitation from infected-cell extracts (μ
NS
). μ
B
represents the major outer capsid protein and is structurally homologous to μ
1C
of the mammalian reoviruses. No additional μ proteins can be detected, and there is no evidence for a product-precursor relationship among these proteins. Three major σ proteins are evident in viral particles. σ
C
has the lowest molecular weight, is part of the outer capsid of the virion, and appears homologous to the mammalian σ
1
protein. Interestingly, it demonstrates the greatest polymorphism of all the polypeptides among the different avian reoviruses examined. σ
B
(36,000 daltons) is a major constituent of the outer capsid and, like σ
C
, is exposed to the surface of the virion. σ
A
(39,000 daltons) appears to be an internal protein. An additional polypeptide band in the σ class having an apparent molecular weight of 34,000 to 35,000 can be seen under three different conditions: (i) in some S1133 reovirus preparations, particularly after prolonged storage, a new band (σ
B′
) appears with a reduction in intensity of σ
B
, suggesting that σ
B′
is a degradation product of σ
B
; (ii) in polypeptides immunoprecipitated from infected-cell extracts, a major band (σ
NS
) is apparent migrating just ahead of σ
B
; (iii) in top component preparations from all avian reoviruses examined, a band (σ
TC
) with mobility identical to that of σ
NS
represents a major constitutent and appears to be incorporated within the particle itself. The relationship among these three bands is not currently known. Avian reovirus polypeptides are thus in general similar to those found in mammalian reoviruses, but differences do exist which may be important for understanding viral structure and assembly.
Coding assignments of the five smaller mRNAs of Newcastle disease virus
Authors: Collins P L , Wertz G W , Ball L A , Hightower L E
Published: January 2020

The polypeptide coding assignments for the five messengers of the 18S size class of Newcastle disease virus (NDV) RNA have been determined by cell-free translation of individual RNAs separated by gel electrophoresis. Listed in order of their decreasing electrophoretic mobilities in acid agarose-urea gels, the coding assignments of the RNAs were as follows: RNA 1, M protein; RNA 2, P protein; RNA 3, NP; RNA 4, F glycoprotein; and RNA 5, HN glycoprotein. RNA 2 also directed the synthesis of 33- and 36-kilodalton proteins, which were tentatively identified as being overlapping segments of the P protein. The 33- and 36-kilodalton polypeptides could be detected in infected cells, but not in purified virions of NDV. Since the other unique NDV RNA, a 35S species, has been shown previously to encode the viral L protein, these results complete the coding assignments of the six known NDV mRNAs.