bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutralizing monoclonal antibodies to Theiler's murine encephalomyelitis viruses
Authors: Nitayaphan S , Toth M M , Roos R P
Published: January 2020

Theiler's murine encephalomyelitis viruses (TMEV) are serologically related picornaviruses which cause both enteric and neurological disease in mice. The biological activities of TMEV vary between the two different TMEV subgroups (TO and GDVII) and with different passage histories of the same TMEV strain (e.g., mouse brain-passed versus tissue culture-passed DA strain of the TO subgroup). We raised neutralizing monoclonal antibodies (mAbs) against tissue culture-passed DA and GDVII strains of TMEV. We produced two mAbs against the DA strain which neutralized all members of the TO subgroup, but not the GDVII subgroup strains (GDVII and FA); these two DA mAbs reacted similarly with both mouse brain-passed DA and tissue culture-passed DA. Of six neutralizing GDVII mAbs, four reacted only to GDVII and FA, whereas two neutralized TO strains as well. These mAbs demonstrate the presence of TMEV group-specific as well as subgroup-specific neutralization and substantiate the division of TMEV into two distinct subgroups. On Western immunoblots one of the two DA mAbs reacted against isolated DA VP1, two GDVII mAbs (which were TMEV group specific) reacted against isolated GDVII VP1 and DA VP1, and the other DA mAb and four other GDVII mAbs required an intact virion conformation for reactivity. An analysis of the epitopes recognized by these mAbs may elucidate sites important in TMEV biological activities.
Recombination between a defective retrovirus and homologous sequences in host DNA: reversion by patch repair
Authors: Schwartzberg P , Colicelli J , Goff S P
Published: January 2020

The genomes of mammalian species contain multiple copies of sequences homologous to exogenous retroviruses. When a mutant retrovirus carrying a lethal deletion in an essential viral gene was introduced into mammalian cells, revertant viruses appeared and spread throughout the culture. Analysis of one such revertant showed that the mutation had been repaired by homologous recombination with endogenous sequences. Our results suggest that defective retroviruses can draw upon the genetic complement of the host cell to repair lesions in viral genes.
Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3' end of the viral mRNA leader sequence
Authors: Budzilowicz C J , Wilczynski S P , Weiss S R
Published: January 2020

cDNA clones that represent various portions of the coronavirus mouse hepatitis virus strain A59 genome RNA have been constructed. cDNAs were synthesized by transcription of genome RNA by using either oligo(dT) or random oligomers of calf thymus DNA as primers. These cDNAs were converted into double-stranded DNA and cloned into pBR322 by standard techniques. The resulting cloned viral DNA fragments were mapped to viral genes by hybridization with Northern blots of intracellular RNA from mouse hepatitis virus strain A59-infected cells. These cDNA clones map in six of the seven viral genes. Clone g344, 1.8 kilobases, is the largest and encompasses gene 5 (which encodes a nonstructural protein) and gene 6 (which encodes the E1 viral glycoprotein) as well as the intergenic regions preceding genes 5, 6, and 7. Sequencing of parts of this cloned DNA show that these three intergenic regions contain a common 11-nucleotide sequence. This sequence shares homology with the 3' end of the viral mRNA leader sequence. Thus, this common intergenic sequence may contain a binding site for a leader RNA that hybridizes to negative-strand viral RNA at the beginning of each gene to prime mRNA synthesis. The different degrees of homology between the leader and its putative binding site may influence the differential rates of transcription of the various viral mRNAs.
Intracellular processing of the Newcastle disease virus fusion glycoprotein
Authors: Morrison T , Ward L J , Semerjian A
Published: January 2020

The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site (Nagai et al., Virology 69:523-538, 1976) into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with [3H] fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.
Propagation of nephropathia epidemica virus in Mongolian gerbils
Authors: Yanagihara R , Goldgaber D , Gajdusek D C
Published: January 2020

Nephropathia epidemica virus, the etiological agent of hemorrhagic fever with renal syndrome in Scandinavia, was serially propagated in Mongolian gerbils (Meriones unguiculatus). Intracerebrally inoculated suckling gerbils developed subclinical infections, with viral antigen found in lung, brain, liver and spleen. The distribution of viral antigen was similar to that seen in experimentally infected bank voles (Clethrionomys glareolus), the principal wild rodent reservoir of nephropathia epidemica virus. This model provides a readily available animal host for the study of experimental nephropathia epidemica virus infection.
Cytomegalovirus early and late membrane antigens detected by antibodies in human convalescent sera
Authors: Middeldorp J M , Jongsma J , The T H
Published: January 2020

Using indirect immunofluorescence and a panel of human convalescent-phase sera, we identified cytomegalovirus (CMV) early and late membrane antigens (CMV-EMA and CMV-LMA, respectively) as separate entities on the surfaces of viable CMV-infected fibroblasts starting at 6 to 12 and 36 to 48 h postinoculation, respectively. For expression of CMV-EMA and CMV-LMA, infectious virus and active protein synthesis were required, whereas the expression of CMV-LMA, in addition, required viral DNA synthesis. Our data suggest that CMV-EMA and CMV-LMA form an individual set of CMV antigens that are different from intracellular CMV antigens and possibly (partly) different from the viral envelope.
Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus
Authors: Makino S , Fujioka N , Fujiwara K
Published: January 2020

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.
Temporal regulation of baculovirus RNA: overlapping early and late transcripts
Authors: Friesen P D , Miller L K
Published: January 2020

Analysis of the temporal sequence of RNA transcription within the Autographa californica nuclear polyhedrosis virus genome revealed individual transcription units composed of overlapping early or late RNAs, or both. High-resolution S1 nuclease mapping within the 3.0-kilobase HindIII-K fragment located five overlapping RNAs (1.07, 1.38, 2.63, 3.16, and 3.50 kilobases) transcribed in the same direction and terminated at a common 3' site. The smallest RNAs appeared early but were replaced in time by successively larger RNAs initiated further upstream. Primer extension analysis supported the contention that this temporal regulation involved the sequential activation of upstream promoters and the coordinate deactivation of downstream promoters. As such, transcription from upstream genes may suppress that of downstream genes via transcriptional interference (promoter occlusion) and thereby facilitate sequential expression of different viral functions. In contrast, overlapping RNAs with extended 3' ends were transcribed from the abundantly expressed p10 and polyhedrin genes mapping to the HindIII-Q,P/EcoRI-P and HindIII-V/EcoRI-I fragments, respectively. These RNAs were synthesized maximally during the very late occlusion phase and consisted of a major small transcript and several larger but less abundant transcripts.
Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM
Authors: Taguchi F , Siddell S G , Wege H , ter Meulen V
Published: January 2020

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.
Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene
Authors: Beisel C , Tanner J , Matsuo T , Thorley-Lawson D , Kezdy F , Kieff E
Published: January 2020

Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M. Hummel, D. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417). These RNAs have the same 5' and 3' ends. The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame. The smaller RNA has one internal splice which maintains the same open reading frame. Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J. Virol. 49:413-417). The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties. Antisera raised in rabbits to the protein expressed in E. coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame. The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement. This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody. We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.
Identification of a new polypeptide coded by reovirus gene S1
Authors: Sarkar G , Pelletier J , Bassel-Duby R , Jayasuriya A , Fields B N , Sonenberg N
Published: January 2020

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).
Synthesis and processing of glycoprotein gG of herpes simplex virus type 2
Authors: Balachandran N , Hutt-Fletcher L M
Published: January 2020

Monoclonal antibody 13 alpha C5-1-A11 immunoprecipitated two major polypeptides of molecular weights 108,000 and 120,000 from extracts of herpes simplex virus type 2-infected BHK-21 cells labeled with [35S]methionine or [3H]glucosamine. In pulse-chase experiments, both labels were chased from the 120,000-molecular-weight peptide (120K peptide) into the 108K molecule. Endoglycosidase H (endo H) reduced the 120K peptide to a 112K peptide but did not affect the 108K peptide. Similar profiles were obtained with monoclonal antibody AP-1 which reacts with a 92K glycoprotein, gG, which maps to the short unique region of the genome. Cross-absorption experiments indicated that both antibodies reacted with the same peptides, suggesting that the 120K peptide is a partially glycosylated high-mannose-type precursor of gG (pgG1). Immunoprecipitation from monensin-treated cells indicated that pgG1(120K) may undergo peptide cleavage to form a 74K high-mannose-type peptide (pgG2) and that this 74K peptide may be further processed into an endo H-resistant 110K to 116K peptide. In the presence of tunicamycin, gG(108K) was replaced by 110K and 105K peptides which were resistant to both endo H and endoglycosidase F. The 105K peptide was the only molecule labeled by [3H]galactose or [3H]glucosamine in the presence of tunicamycin, and none of the peptides were labeled with [3H]mannose, indicating the probable presence of O-linked sugars in the 105K peptide. Our results imply that cotranslational glycosylation of the unglycosylated precursor 110K peptide results in the high-mannose-type pgG1(120K), which probably undergoes peptide cleavage. This putative cleavage product may then mature into gG (108K) by the trimming of sugars and the addition of complex and probably O-linked sugars; the high-mannose-type pgG2(74K) is probably an intermediate peptide formed in this process.
Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses
Authors: Vassef A , Mars M , Dru A , Plucienniczak A , Streeck R E , Beaud G
Published: January 2020

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.
Heterogeneity of the procapsid of bacteriophage T3
Authors: Serwer P , Watson R H , Hayes S J
Published: January 2020

Like other double-stranded DNA bacteriophages, bacteriophage T3 assembles a DNA-free procapsid that subsequently packages the bacteriophage DNA. By agarose gel electrophoresis, it has been found that the T3 procapsid has a negative electrophoretic mobility (mu) that progressively increases in magnitude by as much as 3% after assembly of the procapsid. This increase is (i) caused by an increase in the solid support-free mu (muo) of the procapsid, not a decrease in its radius, and (ii) not prevented by either genetically or chemically (use of proflavine) blocking DNA packaging. However, inhibition of the formation of high-energy compounds with a mixture of cyanide and fluoride ions does block the time-dependent increase in the magnitude of muo. This increase appears to be accompanied by addition of an unidentified T3 protein to the procapsid.
Experimental infection with Puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus)
Authors: Yanagihara R , Amyx H L , Gajdusek D C
Published: January 2020

Subclinical chronic infections characterized by transient viremia, prolonged virus shedding in oropharyngeal secretions and feces, and virus persistence in tissues (particularly lung) developed in laboratory-bred weanling bank voles (Clethrionomys glareolus) inoculated intramuscularly with Puumala virus (strain Hällnäs), the etiologic agent of nephropathia epidemica. Viral antigen, as evidence by granular fluorescence, was detected in the lungs, liver, spleen, pancreas, salivary glands, and small intestine. Infectious virus was found in the lungs from 14 to 270 days postinoculation, and feces and urine collected 35 to 130 days postinoculation were regularly and sporadically infectious, respectively. Horizontal transmission coincided with virus shedding in oropharyngeal secretions. Suckling voles also developed asymptomatic persistent infections after intracerebral inoculation, and histopathological changes were absent despite widespread infection. Our data resemble findings in Apodemus agrarius experimentally infected with Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, suggesting that the mechanisms of maintenance and transmission of Puumala and Hantaan viruses are similar in their respective wild-rodent hosts.
Viral polypeptides detected by a complement-dependent neutralizing murine monoclonal antibody to human cytomegalovirus
Authors: Rasmussen L , Mullenax J , Nelson R , Merigan T C
Published: January 2020

Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000- and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000- and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000- and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35S]methionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfide-linked 130,000-Da polypeptides.
Coevolution of cells and virus as a mechanism for the persistence of lymphotropic minute virus of mice in L-cells
Authors: Ron D , Tal J
Published: January 2020

Infection of L-cells with minute virus of mice (i), a lymphotropic strain of minute virus of mice, resulted in the emergence of host range mutant viruses capable of a lytic infection that destroys the initially restrictive parental cells. Despite that, the culture was not lysed completely; instead, a persistent infection resulted which lasted at least 150 days. Throughout the persistent infection, extensive changes occurred in both the tissue tropism of the progeny virus and in the phenotypic properties of the cells. Mutant cells were selected which were increasingly restrictive to the replication of the resident virus, but concomitant changes in the virus enabled it to replicate in a subpopulation of the restrictive cells. The persistent infection could be reconstructed by infection of mutant cells with mutant virus; in contrast, neither infection of parental cells with mutant virus nor infection of mutant cells with parental virus led to persistence. On the basis of these results, we suggest that virus-cell coevolution provides the primary mechanism for the initiation and the maintenance of the persistent infection.
Potent neutralizing activity associated with anti-glycoprotein D specificity among monoclonal antibodies selected for binding to herpes simplex virions
Authors: Para M F , Parish M L , Noble A G , Spear P G
Published: January 2020

Thirty-three monoclonal antibodies were selected for ability to bind to purified virions of herpes simplex virus and were shown by immunoprecipitation to react with one or another of the envelope glycoproteins. Only six of these antibodies exhibited potent neutralizing activity, and all six were specific for glycoprotein D. Two other anti-glycoprotein D antibodies and 25 antibodies specific for four other viral glycoproteins had much less potent, if any, neutralizing activity.
Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats
Authors: Prince G A , Horswood R L , Chanock R M
Published: January 2020

The amount of passively acquired serum respiratory syncytial virus (RSV)-neutralizing antibodies required to protect the respiratory tract of cotton rats against infection was studied. Infant cotton rats were inoculated intraperitoneally with various dilutions of a single pool of sera derived from cotton rats convalescent from RSV infection. After 24 h, these animals were inoculated with RSV intranasally. Virus replication in the respiratory tract was suppressed in cotton rats which had a serum neutralizing antibody titer of 1:100 or greater. Resistance was greater in the lungs than in the nose. Complete or almost complete resistance in the lungs was observed in cotton rats with a serum neutralizing antibody titer of 1:380 or greater. The level of serum RSV-neutralizing antibodies required to confer significant resistance to infection in the cotton rat was similar to the level of maternally derived serum antibodies possessed by human infants less than 2 months of age, who as a group exhibit relative resistance to RSV disease compared with infants 2 to 6 months of age.
Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products
Authors: Harlow E , Franza B R , Schley C
Published: January 2020

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.
Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies
Authors: Purdy M A , Ghiasi H , Rao C D , Roy P
Published: January 2020

The complete sequence of the RNA which encodes the major outer-shell-neutralizing antigen (VP2) of bluetongue virus serotype 10 was determined from overlapping cDNA clones inserted into pBR322. The segment L2 RNA was 2,926 base pairs long (1.87 X 10(6) daltons) and had, in one strand, an open reading frame capable of coding for a protein that had a calculated size of 111,122 daltons (956 amino acids) and a +11.5 net charge. The coding strands of both the L2 gene and the group-specific L3 gene of bluetongue virus serotype 17 (M. Purdy, J. Petre, and P. Roy, J. Virol. 51:754-759, 1984) had common sequences of some six nucleotides at their 5' termini (namely, GUUAAA...) and eight nucleotides (namely, ...ACACUUAC) at their 3' termini. Both had short 5' noncoding regions with AUG codons at residues 20 to 22 (L2) and 18 to 20 (L3). The sequences flanking these AUG codons were similar (A/GCCAUGG). The 3' noncoding regions were longer (36 nucleotides for L2, 49 nucleotides for L3). The predicted amino acid sequence of the L2, compared with the similarly sized L3 gene product, was rich in cysteine residues and charged amino acids.
Cell-related sequences in the DNA genome of human cytomegalovirus strain AD169
Authors: Shaw S B , Rasmussen R D , McDonough S H , Staprans S I , Vacquier J P , Spector D H
Published: January 2020

Human cytomegalovirus (HCMV) cloned EcoRI fragments R and b hybridized strongly, under standard high-stringency conditions, to uninfected cellular DNA of human, murine, or sea urchin origin. Less hybridization was detected with fragments, A, C, E, WL(F), WN(H), I, M, O, P, Q, V, c, d, and e. Southern blot analysis of the HCMV-related human DNA localized the major sites of hybridization of HCMV EcoRI fragments R, b, and d to defined regions of the 28S rRNA gene.
Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro
Authors: Dietzschold B , Wiktor T J , Trojanowski J Q , Macfarlan R I , Wunner W H , Torres-Anjel M J , Koprowski H
Published: January 2020

Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.
Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells
Authors: Grubman M J , Morgan D O , Kendall J , Baxt B
Published: January 2020

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.
In vivo and in vitro models of demyelinating disease: JHM virus in the rat central nervous system localized by in situ cDNA hybridization and immunofluorescent microscopy
Authors: Sorensen O , Dales S
Published: January 2020

In situ probing of central nervous system (CNS) tissues has made it possible to associate the presence of JHM virus (JHMV) RNA with individual cells in the rat CNS. The presence of viral RNA was not always associated with antigen expression. The in situ hybridization revealed that cerebellar Purkinje cells and hippocampal neurons were highly susceptible to JHMV infection during either acute or paralytic disease. In the paralytic disease, Purkinje cell neurons frequently contained viral RNA. This observation suggests that these neurons, and perhaps others, may be repositories for JHMV in rats that undergo prolonged infections.
Recombination between nonsegmented RNA genomes of murine coronaviruses
Authors: Lai M M , Baric R S , Makino S , Keck J G , Egbert J , Leibowitz J L , Stohlman S A
Published: January 2020

We have isolated a recombinant virus between the A59 and JHM strains of mouse hepatitis virus, which contain a single species of nonsegmented RNA genome. This recombinant was derived by mixed infection of DBT cells with temperature-sensitive mutants of A59 and JHM at nonpermissive temperature. Viruses recovered at this temperature were screened by oligonucleotide fingerprinting of their genomic RNAs. One recombinant virus, B1, was found to contain mostly A59-derived sequences, but the 3 kilobases at the 5' end of the genomic RNA was derived from JHM. Thus, the crossover point in the B1 genome is located within gene A, which codes for the viral RNA polymerases. The study of the intracellular RNA species of B1 virus revealed that probably all of the virus-specific subgenomic mRNA species contained the body sequences of strain A59 but the leader sequences of JHM. This result indicates that the JHM leader RNA, which differs from the A59 leader RNA, could be fused to the mRNAs of a different virus strain during RNA transcription. Furthermore, B1 virus-infected cells contain an additional subgenomic mRNA species which is transcribed from a new initiation site within gene C, suggesting that the leader RNA could determine the site of initiation for coronavirus mRNAs. These data represent a first report of RNA recombination between viruses, other than picornaviruses, which contain nonsegmented RNA genomes.
Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus
Authors: Rodriguez J F , Janeczko R , Esteban M
Published: January 2020

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.
Poliovirus metabolism and the cytoskeletal framework: detergent extraction and resinless section electron microscopy
Authors: Weed H G , Krochmalnic G , Penman S
Published: January 2020

The association of poliovirus metabolism with the cytoskeleton was investigated. Infected cells were extracted by using the nonionic detergent Triton X-100 in the physiological cytoskeleton buffer. The skeletal framework obtained was examined by transmission electron microscopy of resinless sections. The fibers of the framework were grossly distorted in infected cells. No virions or procapsids were seen but many virus-specific spheroidal bodies were associated with the framework. They had a diameter of 40 to 70 nm, were characterized by a dense core and a translucent periphery, and occurred in strings, often near the remnants of flattened vesicles. These spheres may correspond to virus-synthesizing bodies. The metabolism of poliovirus RNA was shown to be associated with the skeletal framework by pulse-labeling cells with [3H]uridine and measuring the RNA retained on the framework. 20S double-stranded RNA, a form of poliovirus RNA found only in the replication complex, was attached to the skeleton throughout a 60-min pulse-label. 35S single-stranded viral RNA, a form found in virions, in polyribosomes, and in the replication complex, appeared first on the framework but after a few minutes was also found in the soluble cytoplasmic phase, encapsidated in virions. In contrast to viral RNA, viral proteins exhibited a varied association with the skeletal framework. Viral proteins were pulse-labeled with [35S]methionine and chased with unlabeled methionine. Although all of the virus-specific proteins were found, to some extent, in the skeletal fraction, the derivatives of P2 (P2-X and P2-5) and a derivative of P3 (P3-2) showed a preferential association with the skeletal framework. Virions and procapsids, on the other hand, were not associated with the cytoskeleton; both they and their component proteins (P1-VP0, P1-VP1, P1-VP2, and P1-VP3) were found dominantly in the soluble cytoplasmic phase. The pathway of poliovirus assembly can be inferred from the above data. It is different from that found previously for the enveloped vesicular stomatitis virus and may be representative of encapsidated cytoplasmic virus assembly.
Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage
Authors: Duyk G , Longiaru M , Cobrinik D , Kowal R , deHaseth P , Skalka A M , Leis J
Published: January 2020

The avian retroviral pol gene-encoded DNA endonuclease (pol-endo) has been shown to selectively cleave the viral long terminal repeat sequences (LTRs) in single-stranded DNA substrates in a region known to be joined to host DNA during integration (G. Duyk, J. Leis, M. Longiaru, and A.M. Skalka, Proc. Natl. Acad. Sci. USA 80:6745-6749, 1983). The preferred sites of cleavage were mapped to the unique U5/U3 junctions found only in covalently closed circular DNA molecules containing two tandem LTRs. The cuts occurred three nucleotides 5' to the axis of symmetry of the 12-of-15-base-pair nearly perfect inverted repeat which marks the LTR junction. Experiments with double-stranded supercoiled DNA substrates revealed a similar specificity for nicking. Also, the endonuclease associated with the pol cleavage product, pp32, has the same specificity as the alpha beta form. The limits of sequence required for site-selective cleavage near the U5/U3 junction were established with single-stranded DNA substrates. A domain no larger than 44 base pairs allowed site-selective cleavage in each strand in vitro. Recognition of either strand appeared to be independent of the other, and in each case, the critical sequence was asymmetrically distributed with respect to the U5/U3 junction. The predominant contribution was from the U5 domain; this is consistent with its conservation in the LTR sequences of a number of avian sarcoma and leukosis viruses.
Genetic reassortment of mammalian reoviruses in mice
Authors: Wenske E A , Chanock S J , Krata L , Fields B N
Published: January 2020

Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo.
Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome
Authors: Ensinger M J , Weir J P , Moss B
Published: January 2020

Fine structure marker rescue involving the use of subfragments of vaccinia virus HindIII DNA fragments L, J, and H has been used to map the mutations in eight temperature-sensitive mutants of vaccinia virus representing four complementation groups. Comparison of their map locations with the positions of the open reading frames and RNA transcripts that have been mapped within this region has allowed the identification of one or two polypeptides as the temperature-sensitive gene product of each mutant.
Molecular and biological characterization of the endogenous ecotropic provirus of BALB/c mice
Authors: Horowitz J M , Risser R
Published: January 2020

We have isolated two identical molecular clones of the single, endogenous ecotropic provirus of BALB/c mice. The BALB/c clones are approximately 1/10 as infectious as an exogenous proviral clone derived from AKR mice, p623. Transfection of mouse cells with each BALB/c proviral clone yielded XC-negative, N-tropic, ecotropic virus. Cotransfection of subgenomic fragments of p623 and the BALB/c provirus did not increase infectivity to the level observed for p623; however, a 292-base-pair fragment of the p623 env gene was found to rescue XC-plaque formation. Sequence analysis showed that the XC-negative BALB/c provirus differed from the XC-positive AKR-derived provirus at a single nucleotide at the junction of the gp70 and p15E envelope proteins. Extensive sequence analysis of the BALB/c endogenous provirus showed that it differed from the sequence of the AKR-derived provirus at approximately 0.5% of 4,500 sequenced nucleotides. In addition, the BALB/c long terminal repeat contains a single copy of the enhancer-containing sequences that are repeated twice in p623. The limited variation between the ecotropic proviruses of BALB/c mice and AKR mice suggests that few cycles of reverse transcription separate these viral genomes.
Cellular DNA regions involved in the induction of rat thymic lymphomas (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc) represent independent loci as determined by their chromosomal map location in the rat
Authors: Tsichlis P N , Lohse M A , Szpirer C , Szpirer J , Levan G
Published: January 2020

The induction of thymic lymphomas by Moloney murine leukemia virus in the rat is linked to provirus integration in at least four independent cellular DNA regions (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc). Because sequences homologous to at least three of these regions (Mlvi-1, Mlvi-2, and c-myc) map to chromosome 15 in the mouse, the question was raised whether they are closely linked in the rat genome and whether provirus integration in any one of these regions affects the same functional domain in rat DNA. In this study, we identified the chromosomal map location of Mlvi-1, Mlvi-2, and Mlvi-3 in the rat by using mouse-rat somatic cell hybrids that lose the rat chromosomes. The results showed that Mlvi-1 maps similarly to c-myc to chromosome 7, and Mlvi-2 maps to chromosome 2. Mlvi-3 probably maps to chromosome 15. We conclude that Mlvi-1, Mlvi-2, and Mlvi-3 are separate and independent genetic loci. Although Mlvi-1 and c-myc map to the same chromosome, they are not related, as determined by hybridization and restriction endonuclease mapping. The chromosomal map location of Mlvi-1 to chromosome 7 and Mlvi-2 to chromosome 2 is interesting, since chromosomal aberrations involving these two chromosomes are reproducibly observed in rat neoplasias induced by a variety of agents.
Nucleotide sequence of a cloned woodchuck hepatitis virus genome: evolutional relationship between hepadnaviruses
Authors: Kodama K , Ogasawara N , Yoshikawa H , Murakami S
Published: January 2020

We have determined the complete nucleotide sequence of a cloned DNA of woodchuck hepatitis virus (WHV), the most oncogenic virus among hepadnaviruses. The genome, designated WHV2, is 3,320 base pairs long and contains four major open reading frames (ORFs) coded on the same strand of nucleotide sequence as in the human hepatitis B virus (HBV) genome. Comparison of the nucleotide sequence and amino acid sequences deduced from it among the genomes of various hepadnaviruses demonstrates that each protein shows an intrinsic property in conserving its amino acid sequence. A parameter, the ratio of the number of triplets with one-letter change but no amino acid substitution to the total number of triplets in which one-letter change occurred, was introduced to measure the intrinsic properties quantitatively. For each ORF, the parameter gave characteristic values in all combinations. Therefore, the relative evolutional distance between these hepadnaviruses can be measured by the amino acid substitution rate of any ORF. These comparisons suggest that (i) the difference between two WHV clones, WHV1 and WHV2, corresponds to that among clones of a HBV subtype, HBVadr, and (ii) WHV and ground squirrel hepatitis virus can be categorized in a way similar to the subgroups of HBV.
Structure and evolution of two related transcription units of Epstein-Barr virus carrying small tandem repeats
Authors: Laux G , Freese U K , Bornkamm G W
Published: January 2020

Two regions of the Epstein-Barr virus (EBV) genome carrying partially homologous clusters of short tandem repeats (NotI and PstI repeats) flanked by 1044 and 1045 base pairs with almost complete homology (DL and DR, left and right duplication, respectively) were most abundantly transcribed into poly(A)+ mRNA after induction with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The nucleotide sequence of both repeat clusters and the conserved upstream regulatory sequences from the M-ABA EBV strain are presented. Nearly the whole part of the sequences coding for the RNAs is covered by the NotI and PstI repeats, respectively. The regulatory sequences for these genes are located in the homologous regions of 1044 and 1045 base pairs (DL and DR, respectively). A CAAT box, a TATA box, and other herpes simplex virus-like elements were identified for both transcription units. The initiation points and the 3' ends of both inducible RNAs were mapped by S1 nuclease analysis. Both genes have open reading frames and may potentially code for proteins with repetitive amino acid compositions. The structure of these two inducible EBV genes is discussed, and an evolutionary model is proposed for the generation of gene duplication in the M-ABA strain of EBV.