bluuebunny/crossref_metadata_2025 huggingface dataset:
A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein
Authors: Cho M S , Milman G , Hayward S D
Published: January 2020

We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.
Interaction between HeLa cells and adenovirus type 2 virions neutralized by different antisera
Authors: Wohlfart C E , Svensson U K , Everitt E
Published: January 2020

Three adenovirus type 2-specified immunogens elicited neutralizing antibodies when injected into rabbits; these were the fiber, the hexon, and the penton base. Adenovirus type 2 virions, neutralized by antihexon- or anti-penton base antisera, attached to HeLa cells to the same extent as untreated control virus, and after attachment, neutralized viruses also became sensitive to DNase treatment. A fraction of 75 to 80% of the attached antibody-treated virions penetrated the plasma membrane, which should be compared with an 84 to 88% penetration level in the control series. A majority of the antihexon-neutralized virions was found in intracellular vesicles, as revealed with an electron microscope, but in the case of anti-penton base neutralization, a maximum of 50% of the virions was retained within vesicles, and ca. 30% was free in the cytoplasmic compartment. A value greater than 45% was never obtained for neutralization with a monospecific anti-penton base antiserum, which could imply the existence of alternative pathways for virus penetration into HeLa cells--one of these being sensitive to treatment with anti-penton base antiserum. Antisera containing antifiber specificities efficiently aggregated virions, and the aggregation data mirrored the degree of neutralization. Antifiber-neutralized virions attached to cells to a three- to five times greater extent than untreated control virus, but the former virions had a reduced ability to become sensitive to DNase treatment. Around 15% of the attached antifiber-treated virions was found as large aggregates inside multivesicular bodies or lysosomes.
Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments
Authors: Sturman L S , Ricard C S , Holmes K V
Published: January 2020

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.
Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus-infected humans
Authors: Meyers M L , Trepo L V , Nath N , Sninsky J J
Published: January 2020

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide. A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of beta-galactosidase was expressed and characterized in bacterial extracts. Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera. The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X. As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).
Comparative analyses of the specificities of anti-influenza hemagglutinin antibodies in human sera
Authors: Wang M L , Skehel J J , Wiley D C
Published: January 2020

Estimates of the variety of specificities of anti-influenza hemagglutinin antibodies in postinfection human sera taken between 1969 and 1971 and in 1978 were made by using Fab fragments of defined monoclonal antibodies in competitive virus-binding assays. The results obtained with the sera taken between 1969 and 1971 indicated that different sera contained antibodies with different ranges of specificities, whereas the 1978 sera mainly contained a broad range of antibodies. The results are discussed in relation to the mechanism of antigenic drift in influenza virus, the commonly observed antigenic heterogeneity of influenza virus isolates, and the efficacy of antiinfluenza vaccination.
Functional interactions of the simian virus 40 core origin of replication with flanking regulatory sequences
Authors: DeLucia A L , Deb S , Partin K , Tegtmeyer P
Published: January 2020

We constructed a matched set of plasmids to investigate the interactions of essential core sequences of the simian virus 40 replication origin with flanking regulatory sequences. Deletions of either T-antigen-binding region I or the 21-base-pair repeated promoter elements reduced replication to 50 to 70% of wild-type levels. The simultaneous deletion of both regions decreased replication to less than 5% of wild-type levels. Thus, the double deletion greatly amplified the defects of the single deletions. We conclude that region I and the 21-base-pair repeats have related rather than independent functions in DNA synthesis. Insertion of a synthetic region I or the adenovirus 2 major late promoter at the late side of isolated core sequences in place of the 21-base-pair repeats failed to restore replication. In contrast, insertion of a single 72-base-pair enhancer element stimulated replication of the core origin more than fivefold. Thus, three distinct regulatory elements appear to facilitate core DNA replication by related mechanisms. Flanking sequences have only a small direct effect on T-antigen binding to naked core DNA. Possible mechanisms of action include the regulation of transcription or of chromatin structure.
Efficient and accurate in vitro processing of simian virus 40-associated small RNA
Authors: Hay N , Amster-Choder O , Aloni Y
Published: January 2020

Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.
Molecular basis of rotavirus virulence: role of gene segment 4
Authors: Offit P A , Blavat G , Greenberg H B , Clark H F
Published: January 2020

Bovine rotavirus NCDV and simian rotavirus SA-11 exhibited markedly different patterns of gastrointestinal tract disease when inoculated orally into newborn mice. A genetic approach was used to define the molecular basis of these differences. The SA-11 strain of rotavirus was more virulent than the NCDV strain when inoculated orally into newborn mice; the dose of SA-11 required to cause diarrhea in 50% of infant mice was 50-fold less than that required for NCDV. Nineteen reassortant viruses were derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant virus double-stranded RNA segments was determined by gene segment migration differences in polyacrylamide gels and hybridization with radioactively labeled parental viral transcripts. The neutralization antigen phenotype of reassortant viruses was determined by plaque reduction neutralization. We found that the dose of SA-11 and NCDV rotavirus required to induce gastroenteritis in newborn mice was determined by gene segment 4. The results suggest that rotavirus virulence may be manipulated by modification or reassortment of gene segment 4.
Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene
Authors: Deen K C , Sweet R W
Published: January 2020

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.
Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses
Authors: Li J P , Bestwick R K , Machida C , Kabat D
Published: January 2020

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.
Cleavage of a viral polyprotein by a cellular proteolytic activity
Authors: Tian Y C , Shih D S
Published: January 2020

The 200,000-dalton polyprotein encoded by the bottom component RNA of cowpea mosaic virus was synthesized in rabbit reticulocyte lysates, and this in vitro-synthesized protein was isolated from the lysate reaction mixture by sucrose density gradient centrifugation. Incubation of the isolated polyprotein with buffer caused no change in the protein, but incubation with reticulocyte lysates or with fractionated lysate proteins resulted in cleavage of the protein into the expected cleavage products (32,000- and 170,000-dalton proteins). This finding indicated that reticulocytes contain a proteolytic activity that is needed for the primary cleavage reaction. A cleavage assay in which we used partially purified preparations showed that cleavage was an ATP-dependent reaction.
Cultivation and characterization of three strains of murine rotavirus
Authors: Greenberg H B , Vo P T , Jones R
Published: January 2020

Three distinct strains of murine rotavirus were adapted to growth in cell culture. These strains are genetically related but not identical; they are serotypically heterogeneous. The cultivatable strains were substantially more infectious (approximately 10(6)-fold) for suckling mice than heterologous simian rotaviruses were. Homologous murine rotavirus strains spread from inoculated to uninoculated litter mates and caused diarrhea, while heterologous rotaviruses did not spread and cause illness.
Characterization and translation of transmissible gastroenteritis virus mRNAs
Authors: Jacobs L , van der Zeijst B A , Horzinek M C
Published: January 2020

Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2.
Heterogeneity in base sequence among different DNA clones containing equivalent sequences of rotavirus double-stranded RNA
Authors: Arias C F , López S , Espejo R T
Published: January 2020

The nucleotide sequences for several complementary DNA clones of the rotavirus genome were determined. When the sequences obtained from different clones for the same regions (16,000 bases) were compared, differences in eight base positions were observed. These discrepancies, approximately 1 in 2,000 bases, may be due to differences in individual RNA genomes resulting from multiple passages; infidelity of DNA synthesis in the cloning procedure; or both factors. Whatever the cause, this frequency of base substitution found in sequences of complementary DNA obtained from the same isolate should be considered when comparing DNA sequences obtained from independent isolates. On the other hand, the frequency of base changes observed suggests that the rotavirus genome is very conserved since the virus used for cDNA synthesis has been continuously passaged for 6 years without plaque purification.
Expression of 100,000-Mr simian virus 40 (SV40) tumor antigen in mouse fibroblasts transfected with replication-defective SV40 genomes
Authors: Verderame M F , Pollack R
Published: January 2020

Simian virus 40 early region mutants which are partially or completely replication defective were tested for their ability to transform postcrisis mouse fibroblasts. All mutants tested were capable of generating anchorage-independent transformants. We have previously reported the presence of a variant tumor antigen of 100,000 Mr (100K protein) generated upon transformation by wild-type simian virus 40 virions which correlates with anchorage-independent growth (Chen et al., Mol. Cell. Biol. 1:994-1006, 1981). In this study, none of the mutants tested produced the 100K variant protein at early (before the fifth) passage. Long-term passage (greater than 20 weeks) permitted the expression of this 100K variant in half of the transformants. Thus the phenotype of these mutants is different from both wild-type simian virus 40 (frequently production of 100K by the third passage, and always by the tenth passage) and the origin-minus class of mutants (no production of 100K at any passage).
Involvement of host DNA gyrase in growth of bacteriophage T5
Authors: Constantinou A , Voelkel-Meiman K , Sternglanz R , McCorquodale M M , McCorquodale D J
Published: January 2020

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.
Demyelinating lesions due to Theiler's virus are associated with ongoing central nervous system infection
Authors: Chamorro M , Aubert C , Brahic M
Published: January 2020

We used in situ hybridization and immunocytochemistry to look for a correlation between virus expression and white matter lesions during late demyelinating disease due to persistent Theiler's virus infection. We found the following. (i) Tissue lesions developed at the site of virus infection. This correlation was not explained by infection of lymphocytes and macrophages. (ii) Large differences in the extent of pathology existed between mice. The amount of inflammation paralleled the number of cells containing viral RNA or viral capsid antigens. (iii) C57BL/6 mice, which are resistant to demyelination, were able to eradicate the infection. Our results are strongly in favor of a mechanism of demyelination in which viral gene products play a central role.
Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice
Authors: Stanley J , Cooper S J , Griffin D E
Published: January 2020

Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.
Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP
Authors: García P , Hermoso J M , García J A , García E , López R , Salas M
Published: January 2020

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.
Structure and processing of the p2 region of avian sarcoma and leukemia virus gag precursor polyproteins
Authors: Pepinsky R B , Mattaliano R J , Vogt V M
Published: January 2020

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.
Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals
Authors: Steimer K S , Higgins K W , Powers M A , Stephans J C , Gyenes A , George-Nascimento C , Luciw P A , Barr P J , Hallewell R A , Sanchez-Pescador R
Published: January 2020

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.
Isolation of a receptor protein involved in attachment of human rhinoviruses
Authors: Tomassini J E , Colonno R J
Published: January 2020

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.
Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences
Authors: Bellini W J , Englund G , Richardson C D , Rozenblatt S , Lazzarini R A
Published: January 2020

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.
Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene
Authors: Longnecker R , Roizman B
Published: January 2020

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.
Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro
Authors: Hey T D , Richards O C , Ehrenfeld E
Published: January 2020

The poliovirus RNA polymerase, 3Dpol, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C. Young, D. M. Tuschall, and J. B. Flanegan, J. Virol. 54:256-264, 1985). Other polyadenylated template RNAs also yielded products that were twice the length of the template. The polarity of the products synthesized from plus-strand poliovirus RNA template was analyzed by Southern blotting using labeled product RNA to probe single-stranded poliovirus DNAs cloned into M13 vectors. The results demonstrated that host factor-mediated polymerase products contain newly synthesized plus-strand sequences as well as the expected minus-strand sequences. Polymerase products primed with oligo(U) were all of minus-strand polarity.
Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses
Authors: Stewart M A , Warnock M , Wheeler A , Wilkie N , Mullins J I , Onions D E , Neil J C
Published: January 2020

Molecular clones of the subgroup A feline leukemia virus FeLV-A/Glasgow-1 have been obtained. Nucleotide sequence analysis of the 3' end of the proviral genome and comparison with the published sequence of FeLV-B/Gardner-Arnstein showed that the most extensive differences are located within the 5' domain of the env gene. Within this domain, several divergent regions of env are separated by more conserved segments. The 3' end of env is highly conserved, with only a single amino acid coding difference in p15env. The proviral long terminal repeats are also highly conserved, differing by only eight base substitutions and one base insertion. Specific probes constructed from the FeLV-A or FeLV-B env genes were used to compare the env genes of various exogenous FeLV isolates and the endogenous FeLV-related proviruses of normal cat DNA. An FeLV-A-derived env probe showed no hybridization to normal cat DNA but detected all FeLV-A and FeLV-C isolates tested. In contrast, an FeLV-B env probe detected independent FeLV-B isolates and a family of endogenous FeLV-related proviruses. Our observations provide strong evidence to support the hypothesis that FeLV-B viruses have arisen by recombination between FeLV-A and endogenous proviral elements in cat DNA.
A second protease of foot-and-mouth disease virus
Authors: Strebel K , Beck E
Published: January 2020

Foot-and-mouth disease virus (FMDV) genes are expressed as a polyprotein which is rapidly processed into the four primary cleavage products L, P1, P2, and P3. In secondary cleavage reactions, these are further processed into the mature proteins. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. For analysis of its biological function, the L gene was mutated by site-directed mutagenesis of cloned cDNA. In vitro translation of in vitro transcripts of these DNAs and expression studies in Escherichia coli showed that the L mutants affect the processing of the viral polyprotein. The mutants isolated were partially or totally defective in processing the polyprotein at the L/P1 junction. These mutants could, however, be processed in the presence of the wild-type L protein. Furthermore, an antiserum directed against the L protein inhibited processing at the L/P1 cleavage site, so that the release of the L protein from the polyprotein was blocked. These data reveal that the L gene product represents a viral protease which catalyzes its own release from the nascent polyprotein.
Chromosomal locations of members of a family of novel endogenous human retroviral genomes
Authors: Horn T M , Huebner K , Croce C , Callahan R
Published: January 2020

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes (T. I. Bonner, C. O'Connell, and M. Cohen, Proc. Natl. Acad. Sci. USA 79:4709-4713, 1982; M. A. Martin, T. Bryan, S. Rasheed, and A. S. Khan, Proc. Natl. Acad. Sci. USA 78:4892-4896, 1981). The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes (R. Callahan, W. Drohan, S. Tronick, and J. Schlom, Proc. Natl. Acad. Sci. USA 79:5503-5507, 1982; I. M. Chiu, R. Callahan, S. R. Tronick, J. Schlom, and S. A. Aaronson, Science 223:364-370, 1984). Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome (R. Callahan, I. M. Chiu, J. F. H. Wong, S. R. Tronick, B. A. Roe, S. A. Aaronson, and J. Schlom, Science 228:1208-1211, 1985). We show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17.
Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins
Authors: Tsukamoto A S , Ponticelli A , Berk A J , Gaynor R B
Published: January 2020

Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G0-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo 32P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G0-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.
Group C adenovirus DNA sequences in human lymphoid cells
Authors: Horvath J , Palkonyay L , Weber J
Published: January 2020

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.
Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence
Authors: Kennedy-Stoskopf S , Narayan O
Published: January 2020

Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma.
Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice
Authors: Kimsey P B , Engers H D , Hirt B , Jongeneel C V
Published: January 2020

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.
Highly infectious plasmids carrying poliovirus cDNA are capable of replication in transfected simian cells
Authors: Kean K M , Wychowski C , Kopecka H , Girard M
Published: January 2020

We examined events leading to production of infectious poliovirus upon transfection of simian cells with plasmids carrying poliovirus cDNA and simian virus 40 transcription and replication signals. The nature of the simian virus 40 promoter upstream from the poliovirus cDNA had no influence on its infectivity. A high specific infectivity was correlated with plasmid replication, dependent on expression of T antigen either encoded by the plasmid or present in host cells (COS-1).
Mutagenesis of the avian erythroblastosis virus erbB coding region: an intact extracellular domain is not required for oncogenic transformation
Authors: Bassiri M , Privalsky M L
Published: January 2020

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus of birds. The AEV-encoded erbB polypeptide, a transmembrane glycoprotein bearing an N-terminal domain exposed on the surface of virally transformed cells, plays a crucial role in AEV-mediated oncogenesis. We report here a characterization of a mutated form of the AEV erbB protein which lacks over two-thirds of the extracellular region of this oncogenic protein. This mutant v-erbB protein, although lacking the three possible extracellular sites of N-linked protein glycosylation, appears unimpaired in the ability to transform cells to an oncogenic phenotype.
Isolation of mycoplasma virus L2 insertion variants and miniviruses
Authors: Dybvig K , Sladek T L , Maniloff J
Published: January 2020

We isolated two spontaneous variants of mycoplasma virus L2. Both variants, designated L2ins1 and L2ins2, contained a 3.1-kilobase-pair (kbp) insertion in the 11.8-kbp wild-type L2 genome. The insert DNA was shown to be derived from two noncontiguous regions of the L2 genome, and L2ins1 and L2ins2 differed only in the location of the 3.1-kbp insertion. We also isolated L2 miniviruses from serial passages of L2, L2ins1, and L2ins2 viruses. Miniviruses contained circular DNA molecules of 3.1 kbp or multimers of 3.1 kbp. Minivirus 3.1-kbp DNAs had the same sequences as the 3.1-kbp insert DNAs found in L2ins1 and L2ins2 viruses. Miniviruses were not infectious and interfered with the growth of L2, L2ins1, and L2ins2 viruses; hence, L2 miniviruses appeared to be defective interfering particles.