bluuebunny/crossref_metadata_2025 huggingface dataset:
LPP-1 Infection of the Blue-Green Alga
Plectonema boryanum
Authors: Sherman Louis A. , Haselkorn Robert
Published: January 2020

The structural proteins of mature LPP-1 particles and the patterns of protein synthesis after LPP-1 infection have been examined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Structural proteins account for 35% of the LPP-1 genome, and proteins that would require about 65% of the total coding capacity have been detected after infection. The major head proteins have molecular weights of 39,000 and 13,000, whereas the major tail protein is an 80,000-molecular-weight species. Host protein synthesis is depressed soon after infection and appears to be entirely shut off by 5 hr. Three classes of viral proteins are distinguished in infected cells, based on their time course of synthesis and their presence in mature virions.
Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat
Authors: Wellinger R J , Garcia M , Vessaz A , Diggelmann H
Published: January 2020

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.
Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein
Authors: Lieberman P M , O'Hare P , Hayward G S , Hayward S D
Published: January 2020

We identified an Epstein-Barr virus (EBV) gene product which functions in transient-expression assays as a nonspecific trans activator. In Vero cells, cotransfection of the BglII J DNA fragment of EBV together with recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene gave up to a 100-fold increased expression of CAT activity over that in cells transfected with the recombinant CAT constructs alone. The BglII J fragment acted promiscuously, in that increased CAT synthesis was observed regardless of whether the promoter sequences driving the CAT gene were of EBV, simian virus 40, adenovirus, or herpes simplex virus origin. Cleavage of cloned BglII-J plasmid DNA before transfection revealed that activation was dependent upon the presence of an intact BMLF1 open reading frame. This was confirmed with subclones of BglII-J and with hybrid promoter-open reading frame constructs. This region of the genome is also present in the rearranged P3HR-1-defective DNA species, and defective DNA clones containing these sequences produced a similar activation of CAT expression in cotransfection experiments. The heterogeneous 45-60-kilodalton polypeptide product of BMLF1 may play an important regulatory role in expression of lytic-cycle proteins in EBV-infected lymphocytes.
Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma
Authors: Riedel N , Hoover E A , Gasper P W , Nicolson M O , Mullins J I
Published: January 2020

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.
Cell receptors for the mammalian reovirus: reovirus-specific T-cell hybridomas can become persistently infected and undergo autoimmune stimulation
Authors: Matsuzaki N , Hinshaw V S , Fields B N , Greene M I
Published: January 2020

We have previously described the development of virus-specific helper T cell hybridomas which recognize structural determinants shared by type 1 and type 3 reoviruses that have been exposed to UV radiation. We have found that T-cell hybridomas become persistently infected with live type 3 reovirus used for the immunization. Persistently infected T-hybridoma cells were found to spontaneously produce interleukin 2 (IL-2). To analyze the mechanism of induction of IL-2 secretion of persistently infected T-cell hybridomas, we exposed T-cell hybridomas specific for UV-treated virus to replicating type 3 reovirus. The T-cell hybridomas became infected but did not produce IL-2 unless simultaneously exposed to syngeneic I-A+ antigen-presenting cells. In this situation, the persistently infected T-cell hybridomas produced IL-2 without being reexposed to virus. This process was not a consequence of nonspecific IL-2 gene activation, which occurs in cells persistently infected with reovirus, because reovirus infection did not activate IL-2 secretion in T-cell hybridomas with other antigenic specificities. Reovirus exposure also resulted in persistent infection of certain antigen-presenting B-cell tumor lines. The persistently infected B-cell tumor lines could stimulate reovirus-specific helper T cells but not T-cell hybridomas of other specificities. The data support the thesis that persistent infection of reovirus-specific T cells creates a mechanism in which the virus released from these cells is processed and then reexpressed by I-A+ antigen-presenting cells. The IA antigen and reovirus structures on the antigen-presenting cells then restimulate the T cells through their specific receptors, resulting in IL-2 synthesis and release. These observations may be relevant to mechanisms of autoimmunity induced by virus.
Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli
Authors: Hu S C , Court D L , Zweig M , Levin J G
Published: January 2020

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).
Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of repeated sequences downstream of T clusters
Authors: Yuen L , Moss B
Published: January 2020

Analysis of the 5' ends of six apparently full-length cloned cDNA copies of the vaccinia virus growth factor gene suggested precise transcriptional initiation at the first purine following a run of five pyrimidines in the noncoding strand. By contrast, the 3' ends exhibited heterogeneity and were distributed over a 46-base-pair region. In each of the six cDNAs, the nucleotide immediately preceding the retained copy of the poly(A) tail corresponded to the first or second T of a TATGT repeat. Clusters of Ts occurred upstream of the 3' ends, but the AATAAA polyadenylation consensus sequence of higher eucaryotes was absent. Determination of the complete sequence of one cDNA revealed exact correspondence with the vaccinia virus growth factor gene, indicating the absence of internal RNA processing.
Identification of coding regions for various Epstein-Barr virus-specific antigens by gene transfer and serology
Authors: Takada K , Shimizu N , Oguro M , Ono Y
Published: January 2020

Baby hamster kidney cells were transfected with BamHI fragments of Epstein-Barr virus (EBV) DNA (B95-8 strain) cloned into the pLTR vector containing retroviral enhancer and promoter sequences. Seventeen fragments (BamHI-A, -B, -C, -D, -E, -G, -K, -L, -M, -O, -P, -Q, -R, -U, -V, -X, and -Z) expressed antigenically distinct EBV-specific products recognized by EBV-immune human sera.
Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants
Authors: Bhat B M , Wold W S
Published: January 2020

Region E3 of adenovirus encodes about nine overlapping mRNAs (a to i) with different spliced structures and with two major RNA 3' end sites termed E3A and E3B. Synthesis of E3 mRNAs was examined by the nuclease-gel procedure at early and late stages of infection by wild-type virus (rec700) and by several E3 deletion mutants. Our results, together with those obtained by electron microscopy (L. T. Chow, T. R. Broker, and J. B. Lewis, J. Mol. Biol. 134:265-303, 1979), suggest that E3 may be differentially regulated at early and late stages at both the promoter and RNA-processing levels. Early after infection, the E3 promoter is used to make mainly mRNAs a and h. Late after infection, the E3 promoter appears to be shut off and the major late promoter is used to make mainly mRNAs d and e. The late L4 mRNA 3' end site is not used early even though early E3 pre-mRNAs transcribe through the L4 RNA 3' end site. The nucleotide 768-951 exon, which is the y leader on many L5 mRNAs, is very abundant late. (Nucleotide +1 is the major E3 transcription initiation site.) Early after infection, the 951 5' splice site is enhanced 5- to 10-fold in the dl712 (delta 1691 to 2122) group of mutants; late after infection, these mutants resemble the wild type. We speculate that the activity of the 951 5' splice site is regulated at early and late stages; it is suppressed early to permit synthesis of mRNA a, and it is activated late to permit synthesis of mRNAs d, e, and L5. With dl719 (delta 2173 to 2237), the 951----2157 splice is enhanced both early and late; we suggest that this deletion enhances the 2157 3' splice site.
Two-Dimensional Polyacrylamide Gel Analysis of
Plodia interpunctella
Granulosis Virus
Authors: Russell Darcy L. , Consigli Richard A.
Published: January 2020

The structural polypeptides of purified
Plodia interpunctella
granulosis virus were analyzed by three different two-dimensional gel systems. Isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of 53 acidic polypeptides in the enveloped nucleocapsid of the virus ranging in molecular weight from 97,300 to 8,000. Nine of these polypeptides were shown to be glycoproteins by the technique of radiolabeled lectin blotting. Separation of the granulin in this system allowed resolution of five species, all of which have identical tryptic peptide maps. This matrix protein was demonstrated to be a phosphoglycoprotein by radiolabeled lectin blotting and acid phosphatase dephosphorylation. Nonequilibrium pH gel electrophoresis followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of the major basic protein of the virus, VP12, from a more acidic protein of the same molecular weight. Tryptic peptide analysis demonstrated that these two proteins were indeed different and acid urea gels followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed localization of the acidic protein to the envelope and the basic protein to the nucleocapsid of the virus. Finally, probing of the separated envelope nucleocapsid proteins in both the isoelectric focusing and nonequilibrium pH gel electrophoresis two-dimensional systems after transfer to nitrocellulose with iodinated, purified viral proteins allowed further insight into reactions which may be important in the maintenance of the virion structure.
Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes
Authors: Kari B , Lussenhop N , Goertz R , Wabuke-Bunoti M , Radeke R , Gehrz R
Published: January 2020

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.
Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region
Authors: Parks G D , Duke G M , Palmenberg A C
Published: January 2020

All picornaviral peptides are derived by progressive posttranslational cleavage of a giant precursor polyprotein. Translation of encephalomyocarditis virus (EMC) RNA in rabbit reticulocyte extracts produces active viral peptides, including protease 3C, which is responsible for many cleavage reactions within the processing cascade. DNA plasmids containing 5' noncoding sequences of EMC linked to other portions of the viral genome were constructed and transcribed into RNA. Like virion RNA, the clone-derived transcripts directed efficient protein translation in vitro. The 5'-linked constructions may represent examples of a general method for cell-free expression of any cloned gene segment. One construction produced a self-cleaving P3 region precursor, which contained active 3C protease. A genetically engineered insertion within the 3C sequences eliminated endogenous self-cleavage activity without altering the ability of the P3 peptide to serve as substrate in bimolecular reactions with added 3C. Another plasmid encoding the L-VP0 portion of the capsid region was used to demonstrate that scission between the leader peptide (L) and capsid protein VP0 can be catalyzed by 3C. The enzyme responsible for this step was previously unidentified. A rapid purification scheme for isolation of 3C from EMC-infected HeLa cells is also presented.
Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV-III/LAV and other lentiviruses
Authors: Benveniste R E , Arthur L O , Tsai C C , Sowder R , Copeland T D , Henderson L E , Oroszlan S
Published: January 2020

A retrovirus has been isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma in 1982 at the University of Washington primate center. This isolate, designated MnIV (WPRC-1) (M. nemestrina immunodeficiency virus, Washington Primate Research Center) shows the characteristic morphology of a lentivirus and replicates to high titers in various lymphocyte lines of human and primate origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MnIV revealed multiple bands of structural proteins, including a major viral gag protein of 28 kilodaltons, that did not comigrate with the viral proteins of a human immunodeficiency virus (HIV [FRE-1]) that was also isolated on HuT 78 cells. The relatedness of MnIV to other lentiviruses (HTLV-III/LAV, EIAV, and visna) was examined in radioimmunoassays, by immunoblot techniques, and by N-terminal amino acid sequence analysis of the viral p28 gag protein. The immunoassays revealed cross-reactivity only between MnIV p28 and HTLV-III/LAV p24, and sequence analysis showed that 14 of the 24 N-terminal residues of MnIV p28 and HTLV-III/LAV p24 are identical. These results indicate that MnIV belongs to the same lentivirus family as HTLV-III/LAV but is only partially related to these human acquired immune deficiency syndrome retroviruses.
Latent infection of KB cells with adeno-associated virus type 2
Authors: Laughlin C A , Cardellichio C B , Coon H C
Published: January 2020

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.
Ion etching of bacteriophage lambda: evidence that the right end of the DNA is located at the outside of the phage DNA mass
Authors: Brown J C , Newcomb W W
Published: January 2020

Bacteriophage lambda was etched in an Ar+ plasma under conditions in which the capsid and some of the DNA were eroded (by sputtering) from the particle surface. Analysis of the DNA remaining in etched phage demonstrated an enrichment in sequences derived from the left end and middle of the genome; sequences from the right end were selectively lost. The results suggest that the DNA in the mature phage is arranged with its left end toward the center and its right end toward the exterior of the overall DNA mass. Since the left end is the first to enter the phage prohead, the results are most compatible with the view that prohead filling also proceeds from the center to the exterior of the cavity. The suggested arrangement of lambda DNA is comparable to that observed in phage T4 and is consistent with the spiral-fold model of packaged DNA.
Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for adeno-associated virus helper activity
Authors: Quinn C O , Kitchingman G R
Published: January 2020

We generated four point mutations in the DNA-binding protein (DBP) gene of adenovirus type 5 by oligonucleotide-directed site-specific mutagenesis. The sites mutated were in the three conserved regions (CR; amino acids 178-186 [CR1], 322-330 [CR2], and 464-475 [CR3]) identified previously by comparative sequence analysis (G. R. Kitchingman, Virology 146:90-101, 1985). The mutations resulted in changes in amino acids 181 (Trp to Leu), 323 (Arg to Leu), 324 (Trp to Leu), and 469 (Phe to Ile). The mutated DBP genes were put under the control of the simian virus 40 early promoter and analyzed by transfection for their ability to help adeno-associated virus replicate its DNA in COS-1 monkey cells. Mutations in the aromatic amino acids 324 and 469 reduced the amount of AAV DNA replication approximately 10-fold, while the mutation in Arg 323 produced a reduction of approximately fourfold. The Trp-to-Leu mutation in amino acid 181 had no effect on AAV DNA replication. The decreased helper activity of the 323, 324, and 469 mutations was not caused by any effect of the mutation on the stability of the DBP. These results suggest that CR2 and CR3 are involved in AAV helper activity, specifically in AAV DNA replication. The relevance of these findings to the identification of residues important for the functions of DBP in adenovirus infection is discussed.
Genetic background affects integration frequency of ecotropic proviral sequences into the mouse germ line
Authors: Bautch V L
Published: January 2020

Germ line acquisition of ecotropic proviruses occurs at a high frequency in the progeny of SWR/J-RF/J hybrid mice carrying two genetically linked RF/J ecotropic proviral loci, Emv-16 and Emv-17 (N. A. Jenkins and N. G. Copeland, Cell 43:811-819, 1985). To determine if genetic background affects proviral integration frequency, I analyzed a series of crosses in which the two RF/J proviral loci were transferred onto different provirus-negative background strains. Unlike SWR/J-RF/J hybrid progeny, few CBA/CaJ-RF/J hybrid mice were identified that carried new germ line proviral loci. These results indicate that genetic factors other than the linked RF/J proviral loci contribute to the increased frequency of germ line provirus integration seen in the SWR/J-RF/J hybrids. The frequency of proviral acquisition appeared to increase when females carrying Emv-16, Emv-17, and at least one new proviral locus were further backcrossed, suggesting that integration frequency can be increased by genetic manipulation. The breeding data are consistent with the hypothesis that virus from the mother infects the egg or the early embryo. Analysis of the transmission frequency and cosegregation patterns of new proviral loci indicated that viral integration occurs after the first round of DNA replication and before the germ line is set aside during embryogenesis, with a majority of viral integrations occurring at the two-cell stage of development, and independent viral integrations can occur in the same or in different cells of the embryo.
Genomic organization of the related Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6
Authors: Santos M A , Almeida J , de Lencastre H , Morelli G , Kamke M , Trautner T A
Published: January 2020

The genomes of the related virulent Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6 are partially circularly permuted and terminally redundant. Heteroduplex molecules were produced with various combinations of these DNAs. Their electron-microscopic analyses showed a consistent pattern of homologous and heterologous regions of DNA. Restriction maps of the phage DNAs were established. A comparison of these maps showed a pattern of conserved and variable DNAs compatible with the electron-microscopic analyses. In all phage genomes, regions specifying early and late functions were conserved. In each phage genome, such regions were separated by short segments of heterologous DNA characteristic for each phage.
A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state
Authors: Lusky M , Botchan M R
Published: January 2020

A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids.
Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product
Authors: Balduzzi P C , Chovav M , Christensen J R , Macara I G
Published: January 2020

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.
Quaternary structure of influenza virus hemagglutinin after acid treatment
Authors: Doms R W , Helenius A
Published: January 2020

Hemagglutinin (HA), a trimeric spike glycoprotein of influenza virus, mediates fusion between the viral envelope and the membrane of an endosome during virus entry. Fusion is triggered by low pH, which induces an irreversible conformational change in the protein. Several studies have indicated that intersubunit contacts along the trimer interfaces may be broken during this alteration. To determine whether HA dissociates into individual subunits as a consequence of the conformational change, we used velocity gradient sedimentation in the presence of Triton X-100. We also determined the resistance of acid-treated HA to dissociation by sodium dodecyl sulfate, a property of the HA trimer. At pH 7.0, isolated HA sedimented as a 9S trimer and gave the characteristic trimer pattern after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After acidification the HA remained trimeric irrespective of whether it was exposed to acid in intact virus particles or in solubilized form. Only when very low concentrations of HA were acidified did a fraction dissociate to dimers and monomers. In contrast, the water-soluble ectodomain fragment of HA (BHA) readily dissociated under a variety of conditions. Negative-stain electron microscopy supported the notion that HA molecules in virus particles do not dissociate upon acidification and may form larger oligomeric structures in the plane of the viral membrane. Taken together, the results suggested that it is the trimeric HA, or higher-order structures thereof, that are active in the acid-induced fusion reaction. Further, the results emphasized the role of the transmembrane anchors of HA in preventing dissociation of the trimer.
Biochemical properties of p60v-src mutants that induce different cell transformation parameters
Authors: Jove R , Garber E A , Iba H , Hanafusa H
Published: January 2020

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.
Identification of two epitopes in the carboxyterminal 15 amino acids of the E1 glycoprotein of mouse hepatitis virus A59 by using hybrid proteins
Authors: Tooze S A , Stanley K K
Published: January 2020

cDNA fragments coding for the carboxy terminus of the E1 envelope glycoprotein from mouse hepatitis virus A59, a coronavirus, were cloned into the bacterial expression vector pEX. Clones expressing E1 antigenic determinants were selected with a polyclonal anti-E1 antibody and used for immunization of rabbits and for affinity purification of existing polyclonal antisera. Immunofluorescence testing and immunoperoxidase labeling of coronavirus-infected cells showed that these reagents were monospecific for E1. In addition, by using hybrid proteins containing different lengths of the E1 carboxy terminus to affinity-purify a polyclonal antiserum against E1, we have been able to define two epitopes within the last 15 amino acid residues of the protein. These epitope-specific antibodies bind to E1 in Golgi and perinuclear membranes as well as to budding viruses; they do not, however, label the plasma membrane or the membranes of post-Golgi vesicles transporting virions to the cell surface.
Construction and characterization of hybrid polyomavirus genomes
Authors: Chuke W F , Walker D L , Peitzman L B , Frisque R J
Published: January 2020

Several studies have suggested that certain unique features of the JC virus (JCV) regulatory region are responsible for the restricted lytic and transforming activities of this virus in vitro. To pursue this possibility, we have constructed hybrid polyomavirus genomes by exchanging the regulatory sequences of JCV, BK virus (BKV), and simian virus 40 (SV40). The host range of JCV was not expanded by the substitution of the BKV or SV40 regulatory signals; such hybrids were nonviable even in primary human fetal glial cells, the sole permissive cell for JCV. However, chimeric DNAs containing JCV regulatory sequences and BKV- or SV40-coding sequences were lytically active, indicating that the BKV and SV40 T proteins were capable of effectively interacting with the JCV replication and transcription signals to yield infectious hybrid viruses. Although JCV regulatory sequences and coding sequences both contributed to the restricted lytic activity of this virus, it appears that the latter sequences, most likely hose encoding the T protein, have a greater influence on this behavior.
Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens
Authors: Thierry F , Heard J M , Dartmann K , Yaniv M
Published: January 2020

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.
Receptor-binding characteristics of monoclonal antibody-selected antigenic variants of influenza virus
Authors: Underwood P A , Skehel J J , Wiley D C
Published: January 2020

Erythrocytes modified to different extents with periodate were used in hemagglutination assays to investigate the binding properties of antihemagglutinin monoclonal antibody-selected antigenic variants of X-31 influenza virus. The results allowed differentiation of groups of variants and are discussed in relation to the nature of the amino acid substitutions in the variant hemagglutinins and their molecular locations relative to the receptor-binding site.
Antisera to scrapie-associated fibril protein and prion protein decorate scrapie-associated fibrils
Authors: Merz P A , Kascsak R J , Rubenstein R , Carp R I , Wisniewski H M
Published: January 2020

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.
Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr
Authors: Okamoto H , Imai M , Tsuda F , Tanaka T , Miyakawa Y , Mayumi M
Published: January 2020

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.
Novobiocin and coumermycin A1 inhibit viral replication and the reactivation of herpes simplex virus type 1 from the trigeminal ganglia of latently infected mice
Authors: Spivack J G , O'Boyle D R , Fraser N W
Published: January 2020

Herpes simplex virus type 1 was reactivated from the trigeminal ganglia of latently infected mice in a quantitative and time-dependent manner. Novobiocin and coumermycin A1 reversibly inhibited the reactivation of herpes simplex virus type 1. They did not inhibit viral replication in permissive cells (CV-1) but did inhibit replication in cells of neuronal origin (C1300) and acutely infected trigeminal ganglia.
Biomolecular analysis of a defective nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection
Authors: Alfieri C , Joncas J H
Published: January 2020

A virus recovered from the saliva of a child with chronic active Epstein-Barr virus (EBV) infection for 8 years was shown to induce EBV early antigen (EBV-EA) in Raji cells and to be expressed into EBV-EA in fresh EBV-negative peripheral blood leukocytes. However, it did not replicate its DNA. Oropharyngeal epithelial cells scraped from recurrent mouth lesions were similarly positive for EBV-EA. DNA extracted from these cells and digested with BamHI contained a 6-kilobase-pair fragment homologous to BamHI fragment V and B1 EBV DNA probes. Furthermore, Southern blots of the BamHI and EcoRI digests of the DNA extracted from the cell lines of the patient (transformed with EBV strain B95-8) and of her mother (spontaneous) revealed, in addition to the expected BamHI G, H, H2, and B1 fragments used as probes, additional shorter ones of a presumably endogenous defective virus.
Heterogeneity of genome rearrangements in rotaviruses isolated from a chronically infected immunodeficient child
Authors: Hundley F , McIntyre M , Clark B , Beards G , Wood D , Chrystie I , Desselberger U
Published: January 2020

Rotaviruses with genome rearrangements, isolated from a chronically infected immunodeficient child, were adapted to growth in BSC-1 cells. Preparations of viral RNA from fecal extracts showed a mixed atypical rotavirus RNA profile, which was due to the presence of at least 12 subpopulations of viruses grossly differing in genotype. Besides various forms of genome rearrangements involving segment 8-, 10-, and 11-specific sequences, reassortment in vivo was likely to have occurred during the emergence of these viruses. The protein products of viral genomes with various forms of segmental rearrangements seemed to be largely unaltered. Genome rearrangement is proposed to be a third mechanism directing the evolution of rotaviruses.
Reindeer papillomavirus transforming properties correlate with a highly conserved E5 region
Authors: Moreno-Lopez J , Ahola H , Eriksson A , Bergman P , Pettersson U
Published: January 2020

A papillomavirus was isolated from the epithelial layer of a cutaneous fibropapilloma on a Swedish reindeer (Rangifer tarandus). Reindeer papillomavirus (RPV) is morphologically indistinguishable from other papillomaviruses, but the restriction enzyme cleavage pattern of its genome is different. No sequence homology was detected between RPV DNA and the DNAs of bovine papillomavirus type 1 (BPV-1) and avian papillomavirus when hybridization was performed under stringent conditions. However, the RPV genome hybridized to the genome of the European elk papillomavirus and the deer papillomavirus under stringent conditions. A physical map of the RPV genome was constructed, and selected regions of the genome, covering the open translational reading frame (ORF) E5 and part of the E1 and L1 ORFs, were studied by nucleotide sequence analysis. The results made it possible to align the RPV genome with the genome of BPV-1. The E5 ORF of RPV has the potential to encode a 44-amino-acid, exceptionally hydrophobic polypeptide which is very similar to the E5 polypeptides of BPV-1 and deer and European elk papillomaviruses. RPV is oncogenic for hamsters and transforms C127 mouse cells in vitro. Several virus-specific mRNAs were detected in RPV-transformed C127 cells.
Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells
Authors: Embretson J E , Temin H M
Published: January 2020

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.
Activation of mutated simian virus 40 enhancers by amplification of wild-type enhancer elements
Authors: Clarke J , Herr W
Published: January 2020

We show that duplication of any one of three separate simian virus 40 enhancer elements, A, B, or C, can compensate for loss of function in the remaining two. Simian virus 40 revertants containing point mutations within the A and C (dpm16) or B and C (dpm26) enhancer elements contain tandem duplications that include the remaining wild-type element. These simple tandem duplications can create enhancers 25-fold more active than that of the parental mutant. These revertants can arise by illegitimate recombination between heterologous viral genomes. This was demonstrated by the recombinants resulting from a mixed infection with the viruses dpm16 and dpm2, which contain mutations in the A and C elements and the B element, respectively.