bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children
Authors: Yaginuma K , Kobayashi H , Kobayashi M , Morishima T , Matsuyama K , Koike K
Published: January 2020

Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences.
Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2
Authors: Bauer M , Guhl E , Graessmann M , Graessmann A
Published: January 2020

T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation. Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome. To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells. Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I. Furthermore, the revertant cells were resistant to retransformation by SV40. Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated. Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable. However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection. Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state.
An aberrant avian leukosis virus provirus inserted downstream from the chicken c-myc coding sequence in a bursal lymphoma results from intrachromosomal recombination between two proviruses and deletion of cellular DNA
Authors: Nottenburg C , Stubblefield E , Varmus H E
Published: January 2020

A chicken bursal lymphoma, LL6, contains avian leukosis virus DNA integrated 3' of the c-myc coding sequences, unlike all other examined bursal lymphomas, which have integrations 5' to c-myc. To better understand this unusual mutation, we examined a molecular clone containing the LL6 c-myc gene and determined the structure of the proviral insertion by DNA sequencing. Viral DNA begins 575 base pairs downstream of the c-myc coding sequences within the untranslated region, disrupting the use of the normal polyadenylation signal. An internal deletion of the provirus extends from within U3 in the 5' long terminal repeat to within the gp37-coding region of the env gene, disabling virus replication and protein synthesis. Both host-virus boundaries appear normal with respect to the site in viral DNA which is joined to host DNA; both long terminal repeats lack the terminal dinucleotide found in unintegrated DNA. However, in contrast to normal integrations, the six bases of cellular sequence at the 5' junction are not repeated at the 3' junction. The DNA sequences immediately downstream of the LL6 recombinant provirus are not part of the c-myc gene; they originate from the same chromosome as c-myc, but at least 15 kilobases (kb) away. In addition, DNA sequences normally residing 3' of c-myc are deleted in LL6. In summary, these results imply that the LL6 provirus is the result of recombination between two proviruses; that both proviruses were originally downstream of c-myc in the same orientation and separated by at least 15 kb; and that the recombination event was preceded, accompanied, or followed by an internal proviral deletion. No transcript could be detected within a 20-kb region downstream of the LL6 provirus, leaving unresolved the question of whether the additional chromosomal alterations make a specific contribution to LL6 tumorigenesis.
Class II polytropic murine leukemia viruses (MuLVs) of AKR/J mice: possible role in the generation of class I oncogenic polytropic MuLVs
Authors: Evans L H , Malik F G
Published: January 2020

We examined the frequency of occurrence of polytropic murine leukemia viruses (MuLVs) in the spleens and thymuses of preleukemic AKR/J mice from 1 week to 6 months of age and analyzed the genomic RNAs of several polytropic isolates by RNase T1 oligonucleotide fingerprinting. Polytropic MuLVs were first detected in the spleens of 3-week-old mice and preceded the appearance of polytropic MuLVs in the thymus by over 1 month. At 4 months of age and older, nearly all mice expressed polytropic MuLVs in both organs. In contrast to previous studies which have identified class I polytropic MuLVs in AKR/J mice, fingerprint analysis of polytropic MuLVs from both young (3- to 4-week-old) and older (5- to 6-month-old) preleukemic mice indicated that a large proportion of viruses at both ages were class II polytropic MuLVs. All polytropic viruses (five isolates) analyzed from 3- to 4-week-old mice were recovered from spleen cells and were class II polytropic MuLVs. In older preleukemic mice, five of seven isolates were class II polytropic MuLVs and two were class I polytropic viruses. Class I and class II polytropic MuLVs were recovered from both the spleens and thymuses of older preleukemic mice. A detailed comparison of the class I and class II polytropic MuLVs from 5- to 6-month-old mice revealed that the nonecotropic gp70 sequences of most of the class I and class II MuLVs were identical, consistent with a common origin for these sequences. In contrast, the nonecotropic p15E sequences of class I MuLVs were clearly derived from different endogenous sequences than the nonecotropic p15E sequences of the class II MuLVs. The in vitro host ranges of class I and class II polytropic viruses were clearly distinguishable. Examination of the in vitro host range of several isolates suggested that the predominant polytropic viruses initially identified in the thymus (2 to 3 months of age) were class II polytropic viruses. The order of appearance of the class I and class II polytropic MuLVs and the identity of the gp70 oligonucleotides of these MuLVs suggested a model for the stepwise generation of class I polytropic MuLVs involving a class II polytropic MuLV intermediate.
Identification of a nonproductive, cell-associated form of measles virus by its resistance to inhibition by recombinant human interferon
Authors: Carrigan D R , Kabacoff C M
Published: January 2020

Subacute sclerosing panencephalitis (SSPE) is a fatal disease in children and young adults that is caused by persistent infection of the central nervous system (CNS) by a nonproductive, cell-associated form of measles virus. Using an experimental model for SSPE (LEC viral strain in newborn hamsters), we have shown previously that establishment of such CNS infections involves selective elimination from the CNS of productively infected cells by host defensive mechanisms, coupled with the selective sparing of cells carrying nonproductive viral forms. That interferon (IFN) may play a role in this process was suggested by the disappearance of productively infected cells from the CNS tissues prior to the appearance of antiviral antibodies and by the demonstration of cell-associated, IFN-resistant viral variants in the virus stocks that were used. Results of this study support these conclusions by showing that similar IFN-resistant viral variants are present in the HBS strain of SSPE-derived measles virus and that these variants, in the presence of IFN, have properties that are similar to those of naturally occurring cell-associated strains of SSPE viruses, e.g., DR, IP3, and Biken. These IFN-resistant forms of HBS virus were isolated and were shown to maintain their resistance to inhibition by IFN after cloning. However, on removal of IFN, they reverted to productive forms similar to the parental HBS virus. The potential role of such viral forms in the pathogenesis of SSPE is discussed.
Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins
Authors: Ho D D , Sarngadharan M G , Hirsch M S , Schooley R T , Rota T R , Kennedy R C , Chanh T C , Sato V L
Published: January 2020

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.
Splice acceptor site for the env message of Moloney murine leukemia virus
Authors: Lazo P A , Prasad V , Tsichlis P N
Published: January 2020

We report the isolation and sequence of a cDNA clone containing part of the env message of the Moloney murine leukemia virus (MoMuLV). This clone was derived from a rat thymic lymphoma induced by MoMuLV. The AG acceptor site employed in this message is located at position 5490 in the MoMuLV genome. This splice site is detectable at the cDNA level by the creation of a novel SacI restriction site not present in the viral genome. In the -1 to -40 region, this AG acceptor site is preceded by four conserved heptanucleotides (PyXPyTPuAPy) that may function as acceptors for removal of the 5' end of the intron.
Active synthesis of hemagglutinin-specific immunoglobulin A by lung cells of mice that were immunized intragastrically with inactivated influenza virus vaccine
Authors: Chen K S , Burlington D B , Quinnan G V
Published: January 2020

Intragastric inoculation with whole-virion vaccine of inactivated influenza virus resulted in production of hemagglutinin (HA)-specific immunoglobulin A (IgA) and IgG both in lung lavage fluids and in serum samples of mice. HA-specific IgA was the predominant isotypic antibody secreted in the lung lavage fluids (average IgA/IgG ratio, 13:1), whereas HA-specific IgG was the major antibody class in serum (average IgA/IgG ratio, 0.3:1). These responses were similar to the antibody responses stimulated by intranasal infection with live influenza virus. In vitro cultures of lymphoid cells from lungs and Peyer's patches, but not from spleens, in the presence of homologous antigen, from mice vaccinated intragastrically synthesized mostly HA-specific IgA. Mice immunized parenterally with inactivated influenza virus produced only IgG in lung lavage fluids and sera. Cultures of lymphoid cells from their spleens, but not their lungs, synthesized HA-specific IgG upon antigenic stimulation in vitro; neither synthesized IgA. These in vitro cell culture results, as well as the inverse relationship of IgA/IgG ratios in lung lavage fluids and sera, demonstrated that the IgA antibody in lung lavage fluids was actively synthesized locally in the lungs of intragastrically immunized mice. This finding was consistent with the migratory distribution of antigen-primed lymphoid cells from Peyer's patches to distant lymphoid tissue such as lung. Intragastric vaccination conferred protection against intranasal challenge with a lethal dose of virulent virus.
Identification of a novel herpes simplex virus type 1-induced glycoprotein which complexes with gE and binds immunoglobulin
Authors: Johnson D C , Feenstra V
Published: January 2020

We detected a glycoprotein on the surface of cells infected with herpes simplex virus type 1 (HSV-1) which, in conjunction with gE, binds immunoglobulin G (IgG). The novel glycoprotein, which has an apparent molecular mass of 70 kilodaltons and was provisionally named g70, was first detected in extracts of HSV-1-infected cells labeled by lactoperoxidase-catalyzed iodination and precipitated with rabbit sera or IgG and protein A-Sepharose. In subsequent experiments, g70 and gE were coprecipitated from extracts of HSV-1-infected cells labeled with [35S]methionine, [35S]cysteine, or 14C-amino acids. We were unable to precipitate a polypeptide analogous to g70 or gE from extracts of HSV-2-infected cells with rabbit IgG and protein A-Sepharose. Partial proteolytic peptide analysis indicated that g70 is structurally distinct from gE and gI). In addition, g70 was electrophoretically distinct from the HSV-1 Us4 glycoprotein gG. HSV-1 gE, expressed in mouse cells transfected with the gE gene, was not precipitated with rabbit IgG, nor could these cells bind radiolabeled IgG, suggesting that gE alone cannot act as an IgG (Fc) receptor. This result, coupled with the findings that gE and g70 are coprecipitated with IgG and with an anti-gE monoclonal antibody, suggests that gE and g70 form a complex which binds IgG. The electrophoretic mobilities of g70 molecules induced by different strains of HSV-1 differed markedly, arguing that g70 is encoded by the virus and is not a cellular protein induced by virus infection.
Amphotropic proviral envelope sequences are absent from the Mus germ line
Authors: O'Neill R R , Hartley J W , Repaske R , Kozak C A
Published: January 2020

We derived an amphotropic murine leukemia virus (MuLV) type-specific probe for use in Southern blot hybridizations with cloned and genomic DNAs. A 133-base-pair RsaI-RsaI fragment from the 5' env region of the amphotropic viral isolate 4070A was subcloned into M13mp18 and radiolabeled in vitro. The probe detected the proviral DNAs in mink cells infected with seven different amphotropic MuLV isolates. The probe did not cross hybridize with the DNAs of molecular clones of ecotropic, mink cell focus-forming, or xenotropic MuLVs; nor did it anneal to the proviral DNAs of four xenotropic or six mink cell focus-forming viral isolates grown in mink cells. DNAs of 12 inbred laboratory mouse strains and more than 15 different wild mouse species and subspecies were examined for the presence of endogenous amphotropic env-related fragments. Amphotropic env-related sequences were found only in the DNAs of wild mice trapped in southern California in an area previously shown to harbor mice producing infectious amphotropic virus. Restriction enzyme analyses of DNAs from these mice showed that amphotropic sequences were not present as germ line copies but were the result of congenital or horizontal infection or both in this population. The DNAs of 11 various mammalian and avian species, including both natural predators of mice and squabs from the farms with virus-positive mice, lacked amphotropic envelope-related sequences.
Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors
Authors: Nussbaum O , Lapidot M , Loyter A
Published: January 2020

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.
Interferon induction by adenovirus type 12: stimulatory function of early region 1A
Authors: Toth M I , Arya B , Pusztai R , Shiroki K , Beladi I
Published: January 2020

Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce by a UV-sensitive mechanism 10 to 20 times more interferon than other types do. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.
Selective translation initiation on bicistronic simian virus 40 late mRNA
Authors: Grass D S , Manley J L
Published: January 2020

We described previously a simian virus 40 (SV40) mutant, pSVAdL, that was defective in synthesis of the late viral protein VP1. This mutant, which contains a 100-base-pair fragment of adenovirus DNA encompassing the major late promoter inserted in the SV40 late promoter region (SV40 nucleotide 294), efficiently synthesizes agnoprotein, a protein encoded by the leader region of the same mRNA that encodes VP1. When the agnoprotein AUG initiation codon in pSVAdL was mutated to UUG, agnoprotein synthesis was abolished, and VP1 synthesis was elevated to wild-type levels. Because levels of late mRNA synthesis were not affected by this mutation, these results support a scanning model of translation initiation and suggest that internal translational reinitiation does not occur efficiently in this situation.
Processing of vimentin occurs during the early stages of adenovirus infection
Authors: Belin M T , Boulanger P
Published: January 2020

Evidence is presented that a fraction of vimentin, a component of cytoskeleton recently found to be associated with intracytoplasmic, migrating adenovirus type 2 (Ad2), is processed into smaller polypeptides at early times after infection. The extent of vimentin cleavage appears to depend upon both the multiplicity of infection and the adenovirus serotype. Ad2, Ad5, Ad4, and Ad9 induced similar vimentin cleavage in infected cells, whereas Ad3, Ad7, and Ad12, for which most infecting particles are found sequestered within phagosomes, induced very little, if any, vimentin breakdown. This suggests that vimentin processing is in some way related to the number of virus particles migrating through the cytoplasm. Experiments performed in vitro and in vivo with adenovirus temperature-sensitive mutants H2 ts1 and H2 ts112 and UV-inactivated wild-type Ad2 indicated that vimentin processing is due to a nonvirion, cytoskeleton-associated, proteolytic enzyme activated by adenovirus and sharing characteristics with the protease described by Nelson and Traub (W.J. Nelson and P. Traub, J. Cell Sci. 57:25-49, 1982). The activity of this protease appears to be required for productive infection by adenovirus serotypes 2 and 5 (subgroup C), 4 (subgroup E), and 9 (subgroup D) but not by the oncogenic serotypes 3 and 7 (subgroup B) and 12 (subgroup A).
Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus
Authors: Gnann J W , Nelson J A , Oldstone M B
Published: January 2020

Sera from virtually all individuals infected with human immunodeficiency virus contain antibodies against the viral envelope glycoproteins. By using a series of synthetic peptide antigens, we identified an immunodominant domain at amino acid position 598-609 of gp41. The minimal essential epitope is a 7-amino-acid sequence (amino acids 603-609) containing two cysteine residues. Both cysteine residues are required for the antigenic conformation of the sequence, possibly due to creation of a cyclic structure via disulfide bond formation.
Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication
Authors: Gish W R , Botchan M R
Published: January 2020

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.
Assembly and processing of the disulfide-linked varicella-zoster virus glycoprotein gpII(140)
Authors: Montalvo E A , Grose C
Published: January 2020

Varicella-zoster virus (VZV) specifies the synthesis of at least four families of glycoproteins, which have been designated gpI, gpII, gpIII, and gpIV. In this report we describe the assembly and processing of VZV gpII, a structural protein of an apparent Mr of 140,000, which is the homolog of gB of herpes simplex virus. For these studies, we used two anti-gpII monoclonal antibodies which exhibited both complement-independent neutralization activity and inhibition of virus-induced cell-to-cell fusion. Pulse-chase labeling experiments identified a 124,000-Mr intermediate which was chased to the mature 140,000-Mr product when analyzed in nonreducing gels; in the presence of a reducing agent, the native gp140 was cleaved into two closely migrating species (gp66 and gp68). The biosynthesis of VZV gpII was further analyzed in the presence of the following inhibitors of glycoprotein processing: tunicamycin, monensin, castanospermine, swainsonine, and deoxymannojirimycin. All intermediate and mature forms were digested with endoglycosidases H and F, neuraminidase, and O-glycanase to further define high-mannose, complex, and O-linked glycans. Finally, the addition of sulfate residues was investigated. This characterization of VZV gpII revealed the following results. (i) gp128 and gp124 were early high-mannose forms, (ii) gp126 was an intermediate form with complex N-linked oligosaccharides, (iii) gp130 was a later intermediate with both N-linked and O-linked glycans, and (iv) the mature product gp140 contained a mixture of N-linked and O-linked glycans which were both sialated and sulfated. Further investigations indicated that gpII sulfation was inhibited by tunicamycin and castanospermine but not by deoxymannojirimycin or swainsonine. We also concluded that VZV gpII displayed many biological and biochemical properties similar to those of its herpes simplex virus homolog gB.
Characterization of somatically acquired ecotropic and mink cell focus-forming viruses in lymphomas of AKXD recombinant inbred mice
Authors: Mucenski M L , Taylor B A , Copeland N G , Jenkins N A
Published: January 2020

The DNA of lymphomas from 12 AKXD recombinant inbred mouse strains was analyzed to determine the presence of somatically acquired ecotropic and mink cell focus-forming proviruses. Mink cell focus-forming proviruses were associated primarily with T-cell lymphomas, whereas ecotropic proviruses were associated with lymphomas of B-cell and myeloid lineages. A model based on the results is proposed to explain the variation in lymphoma types observed in different AKXD strains.
Localization of calcium on the polyomavirus VP1 capsid protein
Authors: Ludlow J W , Consigli R A
Published: January 2020

Our laboratory has previously shown that the divalent cation Ca2+ is an integral part of the polyomavirus and plays a major role in stabilizing the intact virion structure. In this report, we show that calcium is sequestered on the major capsid protein VP1 of polyomavirus. The virion structural proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose and probed with 45CaCl2. Autoradiography revealed 45Ca binding exclusively to VP1. Increasing the amount of VP1 transferred to the nitrocellulose resulted in a concomitant increase in 45Ca binding. 45Ca binding to VP1 could be reduced by competition with an excess of unlabeled CaCl2. Separation of the species of VP1 by two-dimensional gel electrophoresis before electroblotting and probing with 45CaCl2 revealed that all six species (A to F) bind the radiolabeled calcium. Formic acid cleavage of the 43-kilodalton (kDa) VP1 protein into 29-, 18-, and 16-kDa fragments before 45Ca-binding analysis revealed that only the 18- and 16-kDa carboxyl-terminal fragments of this protein bind 45Ca.
Rotavirus-specific cytotoxic T lymphocytes cross-react with target cells infected with different rotavirus serotypes
Authors: Offit P A , Dudzik K I
Published: January 2020

Splenocytes from adult C57BL6 (H-2b) mice orally inoculated with nonmurine rotaviruses lysed syngeneic rotavirus-infected target cells. Cytotoxic T lymphocytes (CTLs) were responsible for this cytotoxic activity. Cytotoxic activity was (i) detected 7 days after primary oral inoculation; (ii) not detected in uninoculated animals; (iii) specific for rotavirus-infected target cells; (iv) eliminated by treatment with Thy 1.2-specific immunoglobulin M and complement; and (v) restricted at H-2Db. In addition, rotavirus-specific CTLs cross-reacted with target cells infected with different human or animal rotavirus serotypes. Heterotypic protection against rotavirus challenge may be mediated by cross-reactive CTLs.
In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity
Authors: Willey R L , Smith D H , Lasky L A , Theodore T S , Earl P L , Moss B , Capon D J , Martin M A
Published: January 2020

Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.
Molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene
Authors: Arella M , Lavallée C , Belloncik S , Furuichi Y
Published: January 2020

The double-stranded RNA genome of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was converted to double-stranded DNA and cloned into plasmid pBR322. The complete nucleotide sequence of cloned genome segment 10, which encodes virus polyhedrin polypeptide, was determined. The CPV polyhedrin gene consists of 942 based pairs and possesses a long open reading frame that codes for a polypeptide of 248 amino acids (molecular weight, 28,500), consistent with an apparent molecular weight of 28,000 previously determined for purified polyhedrin. No sequence homology was found between CPV polyhedrin and polyhedrins from several nuclear polyhedrosis viruses. In addition to the polyhedrin gene, we completed the sequence analysis of a small deletion mutant gene derived from the polyhedrin gene. This mutant gene consists of two subset domains of the polyhedrin gene, i.e., the 5'-terminal 121 base pairs and the 3'-terminal 200 base pairs. An in vitro transcription demonstrated that the small mutant gene is transcribed by virion-associated RNA polymerases. These data confirm the importance of CPV terminal sequences in virus genome replication.
Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles
Authors: Furlong D B , Nibert M L , Fields B N
Published: January 2020

Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the mu 1C or sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state.
Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster
Authors: Miner J N , Weinrich S L , Hruby D E
Published: January 2020

Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.
Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin
Authors: Brown L E , Ffrench R A , Gawler J M , Jackson D C , Dyall-Smith M L , Anders E M , Tregear G W , Duncan L , Underwood P A , White D O
Published: January 2020

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.
Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation
Authors: Trempe J P , Carter B J
Published: January 2020

We studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p40 promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p40. When the rep gene was present in cis or in trans, cat expression from p40 was decreased 3- to 10-fold, but there was a 2- to 4-fold increase in the level of p40 mRNA. Conversely, cat expression increased and the p40 mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p40 mRNA levels. We also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p5 promoter in a fashion independent of rep.
Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers
Authors: Crum C P , Nuovo G , Friedman D , Silverstein S J
Published: January 2020

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.
Structure and function of endogenous feline leukemia virus long terminal repeats and adjoining regions
Authors: Berry B T , Ghosh A K , Kumar D V , Spodick D A , Roy-Burman P
Published: January 2020

The nucleotide sequence of the 5' long terminal repeat (LTR) of three independent loci (CFE-6, CFE-16, and CF-14) of endogenous feline leukemia virus (FeLV) DNAs of the domestic cat genome was determined. The 3' LTR of the CFE-6 clone was also sequenced. The endogenous FeLV LTRs, which were very similar to each other in sequence and in organization of the functional domains, differed considerably from the exogenous FeLV LTR in the U3 region. The major differences in U3 included variations in sets of small (14 to 19 base pair) direct repeats, altered location of the simian virus 40 core enhancer-like sequence, and occurrence of three segments of largely nonhomologous sequences. There was extensive homology between endogenous and exogenous FeLV LTRs in sequences beginning from the TATA box through the R region down to the 3' end of the U5 region. The DNA sequence downstream of the 5' LTR encompassing the primer-binding site, leader, and almost to the end of the p15gag coding region, a point up to which the sequencing was carried out, also revealed a high degree of conservation. However, the detection of frameshift and nonsense mutations in this region of a nearly full-length endogenous provirus sequence (CFE-6) predicted its defectiveness and correlated with the lack of infectivity of this DNA. The functional studies of the endogenous LTRs, based on linkage to the bacterial cat gene and transient expression in feline cell lines, indicated that although the basic characteristics for promotion and enhancement of transcription were retained in each LTR, there was a significant variation in the activity of the cat constructs. Reconstruction and deletion analyses with the CFE-6 5' LTR revealed the presence of strong transcription regulatory sequences in the 702-base-pair region immediately upstream of the 5' boundary of the endogenous LTR. These and related data suggest that in addition to the transcription-modulating elements occurring within the LTR, the cis-acting nucleotide sequences in the upstream cellular DNA may determine the overall efficiency of transcription of the defective endogenous FeLV provirus loci of the felid genome.
Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells
Authors: Heinzel S S , Krysan P J , Calos M P , DuBridge R B
Published: January 2020

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.
Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection
Authors: Maier K , Gabriel P , Koscielniak E , Stierhof Y D , Wiedmann K H , Flehmig B , Vallbracht A
Published: January 2020

The production of interferon (IFN) during a chromium-51 release assay with hepatitis A virus (HAV)-infected fibroblasts and autologous peripheral blood lymphocytes from patients with acute HAV infection was studied to determine whether IFN plays a role in immunopathogenesis of hepatitis A infection in humans. Skin fibroblasts of eight patients after acute HAV infection and from two control persons without history of current or past HAV infection were infected with HAV. Peripheral blood lymphocytes were collected at different times after the onset of icterus and tested in a chromium-51 release assay against autologous HAV-infected skin fibroblasts for their cytolytic and IFN-producing activity. The IFN produced during the assay was characterized and found to have the properties of human gamma IFN. Cytotoxicity and gamma IFN release were virus specific. The cell types responsible for both functions were characterized and found to be in the HLA-dependent T8+ lymphocyte subset. Considering that gamma IFN has an antiviral effect on persistent HAV infection in vitro and that it probably accounts for stimulation of HLA class I antigen expression on hepatocytes, our experimental results presented here demonstrate that human gamma IFN produced by HAV-specific T cells may participate in pathogenesis of hepatitis A infection in humans.
Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27
Authors: Rice S A , Knipe D M
Published: January 2020

Herpes simplex virus type 1 (HSV-1) encodes several alpha (immediate-early) gene products that modulate gene expression during viral replication. We report here that the alpha protein ICP27 specifically stimulates expression of a later viral gene, that encoding glycoprotein B (gB). Using temperature-sensitive viral mutants, the effect of ICP27 on HSV-1 protein synthesis was examined at early times after infection or at later times when viral DNA replication was inhibited. Under these conditions, the expression of gB showed a marked dependence on the presence of functional ICP27, whereas several other beta and gamma 1 genes showed a lesser dependence. It was also noted that cells infected with ICP27 temperature sensitive mutants at the nonpermissive temperature showed a reduction in the electrophoretic mobility of the alpha protein ICP4. To examine the mechanism by which ICP27 stimulated gB expression, a plasmid was constructed in which the promoter-regulatory region of the gB gene was fused to the gene encoding chloramphenicol acetyltransferase (CAT). CAT expression from this plasmid was induced significantly by ICP27 expressed from a cotransfected plasmid. Induction of CAT activity by ICP27 correlated well with an increase in the amount of CAT transcripts initiated from the transcriptional start site of the gB gene. The transactivating activity of ICP27 was specific for the gB promoter-regulatory region, as expression from several other HSV-1 promoter-CAT chimeric genes was not stimulated by ICP27. The DNA sequences which conferred the response to ICP27 mapped within 175 base pairs upstream and 41 base pairs downstream of the gB transcriptional start site. Our results suggest that the full expression of gB and perhaps other viral genes during HSV-1 infection requires the combined action of multiple viral transactivators.
Enhanced viral resistance in transgenic mice expressing the human beta 1 interferon
Authors: Chen X Z , Yun J S , Wagner T E
Published: January 2020

Recombinant plasmids pMTIF-beta 1A and pMTIF-beta 1B were constructed by fusing the metallothionein I promoter-regulatory region to the human beta 1 interferon (HuIFN-beta 1) gene. These linearized fusion genes were then introduced into mouse germ lines by zygote microinjection. The chromosomal integration and the germ line transmission of the injected DNA sequences in the resulting transgenic mice were detected by DNA dot blot and Southern transfer hybridizations. The sera of at least two strains of metallothionein/HuIFN transgenic mice were found to protect human WISH cells against vesicular stomatitis virus infection, and this activity could be neutralized by preincubation with anti-HuIFN-beta 1 antibody. These transgenic mice demonstrated significantly enhanced resistance to pseudorabies virus compared with nontransgenic mice when inoculated with pseudorabies virus. The level of resistance seemed to correlate with the concentrations of HuIFN-beta 1 in serum. These transgenic mice may be used as models to study IFN-induced responses and may serve as prototypes to generate disease-resistant animals.