bluuebunny/crossref_metadata_2025 huggingface dataset:
Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4
Authors: Olson N J , Marchin G L
Published: January 2020

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly.
Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin
Authors: Parsons S J , McCarley D J , Ely C M , Benjamin D C , Parsons J T
Published: January 2020

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.
Reactivation of persistent papovavirus K infection in immunosuppressed mice
Authors: Greenlee J E , Dodd W K
Published: January 2020

Papovavirus K (K virus) is a murine papovavirus that produces a fatal interstitial pneumonia in newborn mice and a clinically inapparent infection in older animals. The present study was conducted to determine whether the virus produces latent infection in animals surviving acute infection and whether the infection can be reactivated by immunosuppression. Mice were inoculated by the oral route with 100 newborn mouse 50% lethal doses at 12 days of age and followed for 8 months by using immunofluorescence staining. Cells positive for K virus capsid antigen were found in lungs, livers, kidneys, intestines, and brains for 6 months, but not thereafter. Organ examined at 8 months were negative for virus by tissue culture assay, mouse inoculation, explantation, and cocultivation. Immunosuppression of the remaining animals with 8 weekly injections of cyclophosphamide (150 mg/kg) resulted in the reappearance of viral antigen and infectious virus in multiple organs including brains. The highest titers of virus were present in kidneys. One animal sacrificed after 42 days of immunosuppression was found to have a small pulmonary adenoma or alveologenic carcinoma, but efforts to explant this tumor into tissue culture were unsuccessful. The present study demonstrates that K virus produces a latent infection that is reactivated by immunosuppression, and our results raise questions as to whether reactivated infection may occasionally be associated with the development of neoplasia.
Uncoating of influenza virus in endosomes
Authors: Yoshimura A , Ohnishi S
Published: January 2020

The intracellular uncoating site of influenza virus was studied by measuring the fluorescence intensity of probes conjugated to the virus or the isolated hemagglutinin and also by assaying virus replication under various incubation conditions. Acidification of the viral environment was monitored by the decrease in the fluorescence intensity of fluorescein isothiocyanate, and transport of the virus particles into secondary lysosomes was assayed by the increase in the fluorescence intensity of fluorescein isothiocyanate diphosphate. The intracellular pH was estimated by the ratio of fluorescence intensities excited at two different wavelengths. It was found that the viral environment became acidified to a pH value of 5.1 to 5.2 within 10 min at 37 degrees C or 1 h at 20 degrees C after endocytosis. Addition of ammonium chloride to the medium rapidly raised the pH to 6.7. Transport of the virus particles into the secondary lysosomes was slower and negligibly low during those incubation periods. Virus replication occurred when the cells were incubated for 10 min at 37 degrees C or for 1 h at 20 degrees C, followed by incubation in the presence of ammonium chloride for a total of 12 h. These results indicate the uncoating of influenza virus in endosomes before reaching the secondary lysosomes.
Sites of copy choice replication involved in generation of vesicular stomatitis virus defective-interfering particle RNAs
Authors: Meier E , Harmison G G , Keene J D , Schubert M
Published: January 2020

The copy choice model for the generation of defective interfering (DI) particles of vesicular stomatitis virus suggests that during replication the polymerase prematurely terminates, moves with the nascent daughter strand to another site on the same or a different template molecule, and resumes elongation of the nascent chain. We have analyzed the sites where premature termination or resumption of replication has occurred during the generation of the deletion DI particle LT, the snapback DI particle 011, and the panhandle DI particles T, T(L), and 611. The recombination sites were identified by comparing the nucleotide sequences of the relevant regions of these DI particle RNAs to those of the vesicular stomatitis virus L gene (Schubert et al., J. Virol. 51:505-514, 1984). Sequence homology was not detected between these sites, which rules out the existence of a general terminator or promoter sequence involved in copy choice replication. In several cases, however, premature termination or resumption of RNA replication may be favored by specific signal sequences. The sequences immediately before the start and at the end of the deletion in DI LT contain two hexanucleotides, ATCTGA and GATTGG, in a similar spacing. In these case of DI T and 611, but not of DI T(L), the end of the 5'-terminal region bears the hexanucleotide CCUCUU. This sequence is also repeated in the stem region in all three DI particle genomes. In addition, we present data that the added 3'-terminal regions of the panhandle DI particle RNAs may differ by only one base and are 46 [DI T(L) and 611] or 45 (DI T) bases long. We suggest that each site of the vesicular stomatitis virus genome has the potential to give rise to DI particle RNAs. Specific sequences, however, may modulate this process in a quantitative way, and they favor the generation of certain types of DI particle genomes like those of the panhandle type.
Specific interactions of vesicular stomatitis virus L and NS proteins with heterologous genome ribonucleoprotein template lead to mRNA synthesis in vitro
Authors: De B P , Banerjee A K
Published: January 2020

Two dissociable proteins, L and NS, and N-RNA template were purified from two serologically distinct vesicular stomatitis viruses, Indiana [VSV(IND)] and New Jersey [VSV(NJ)]. Requirements for RNA synthesis in heterologous reconstitution reactions in vitro were studied. The L and NS proteins of VSV(NJ) failed to synthesize full-length leader RNA and mRNAs in vitro when reconstituted with N-RNA(IND) template. However, when purified homologous NS(IND) was added to the reaction mixture, mRNA synthesis ensued. The requirements for transcription of N-RNA(NJ) template were different from those for N-RNA(IND). For RNA synthesis, transcription specifically required L(NJ), but the NS(NJ) and NS(IND) proteins were interchangeable. This suggests that there are specific domains on the L(NJ) protein, at which NS proteins of both serotypes may interact to form an active RNA polymerase complex, whereas L(IND) lacked such domains for interaction with NS(NJ). The function of the L protein appeared primarily to initiate RNA chains, and the NS protein was required for chain elongation. The results of these in vitro complementation experiments are discussed in light of previous in vivo complementation studies.
Cloning of the bluetongue virus L3 gene
Authors: Purdy M , Petre J , Roy P
Published: January 2020

The genes of the bluetongue virus (BTV) serotype 17 have been cloned into pBR322 by tailing both strands of the double-stranded RNA with polyadenylic acid, transcribing them with reverse transcriptase with an oligodeoxythymidylic acid primer, hybridizing the cDNA products, and completing them into duplex structures with the Klenow fragment of Escherichia coli DNA polymerase. After cloning the double-stranded cDNA molecules into pBR322, the complete sequence of the cloned L3 gene was determined. The clone is 2,772 nucleotides long (1.78 X 10(6) daltons), excluding the 3' polyadenylic acid sequence, and has an open reading frame which codes for a protein of some 901 amino acids (103,412 daltons). This clone can hybridize L3 RNA segments of three other U.S. BTV serotypes, BTV-10, -11, and -13 in addition to -17 but not the equivalent RNA segment of epizootic hemorrhagic disease virus of deer, an orbivirus related to BTV.
Monoclonal antibody to simian virus 40 small t
Authors: Montano X , Lane D P
Published: January 2020

A monoclonal antibody, PAb280, was produced that recognizes simian virus 40 (SV40) small t but does not react with SV40 large T. The specificity of the antibody was analyzed by immunoprecipitation of labeled cell extracts, Western blotting, and immunocytochemistry. Small t was found to accumulate late in the SV40 lytic cycle and was localized in both the cytoplasm and the nucleus of cells infected with wild-type SV40. Importantly, antibodies against determinants common to SV40 large T and small t did not appear to be able to recognize the cytoplasmic form of SV40 small t at the immunocytochemical level. The localization of small t within the nucleus appeared to be distinct from that of large T.
Differential expression of murine leukemia virus loci in chemically induced hybrid cells
Authors: Kozak C A
Published: January 2020

The time course of murine leukemia virus production after chemical induction was determined in hamster-mouse somatic cell hybrids containing the xenotropic murine leukemia virus induction locus Bxv-1 or the ecotropic locus Akv-2. By using these hybrids, induction could be studied in the absence of secondary virus spread because xenotropic viruses cannot infect hybrid cells and ecotropic viruses cannot infect hybrids which have lost mouse chromosome 5. After induction, hybrids with Bxv-1 produced only a transient burst of virus, whereas those with Akv-2 continued to produce virus for periods in excess of 3 months. The presence or absence of other mouse chromosomes in the hybrid lines did not alter these induction patterns. Thus, endogenous murine leukemia virus loci differ in their response to induction, and both inducibility and the kinetics of virus expression are controlled at or near these proviral loci.
Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies
Authors: Ackermann M , Braun D K , Pereira L , Roizman B
Published: January 2020

Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes simplex virus (HSV) 1 and 2 and by HSV-1 X HSV-2 recombinants indicated that monoclonal antibody H1183 reacted with HSV-1 alpha ICP0, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 alpha ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described (D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these alpha proteins. The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICP0 was considerably more soluble than ICP4 and ICP27. ICP0, ICP4, and ICP27 were readily partially purified by immunoaffinity chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl sulfate. ICP0 and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h) or late (18 to 20 h) postinfection. A fraction, but not all, 32P that was incorporated early was chased in the presence of unlabeled phosphate. ICP0, ICP4, and ICP27 labeled with either 32P or [35S]methionine yielded multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at the origin when the direction of migration was reversed.
Fine-structure analyses of lipid-protein and protein-protein interactions of gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping
Authors: Pepinsky R B , Vogt V M
Published: January 2020

In avian sarcoma and leukemia viruses, the gag protein p19 functions structurally as a matrix protein, connecting internal components with the viral envelope. We have used a combination of in situ cross-linking and peptide mapping to localize within p19 the regions responsible for two major interactions in this complex, p19 with lipid and p19 with p19. Lipid-protein cross-links were localized near the amino terminus within the first 35 amino acids of the polypeptide. Homotypic protein-protein disulfide bridges were found to originate from near the carboxy terminus of p19, from cysteine residues at amino acids 111 and 153. These results suggest that p19 is divided into domains with distinct functions. The peptide maps constructed for p19, and for the related proteins p23 in avian sarcoma and leukemia viruses and p19 beta in recombinant avian sarcoma viruses, should serve as useful tools for other types of studies involving these proteins.
Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel
Authors: Wilhelmsen K C , Eggleton K , Temin H M
Published: January 2020

Reticuloendotheliosis virus strain T (Rev-T) is a highly oncogenic replication-defective retrovirus which contains the oncogene v-rel. It is thought that Rev-T arose when a virus similar to Rev-A, the helper virus of Rev-T, infected a turkey and recombined with c-rel from that turkey. There is one large c-rel locus in the turkey genome which contains all of the sequences homologous to v-rel (K. C. Wilhelmsen and H. M. Temin, J. Virol. 49:521-529, 1984). We have sequenced v-rel and its flanking sequences, each of the regions of the c-rel locus from turkey that are homologous to v-rel and their flanking sequences, and the coding sequence for env and part of pol of Rev-A. The v-rel coding sequences can be translated into a 503-amino acid env-v-rel-out-of-frame-env fusion polypeptide. We have not detected any sequences in the Los Alamos or University of California-San Diego data bases that are more significantly related to the amino acid or nucleic acid sequence of v-rel than to the randomized sequence of v-rel. Comparison of Rev-A, Rev-T, and c-rel indicates that the v-rel sequences may have been transduced from the c-rel (turkey) locus by a novel mechanism. There are sequences in Rev-A and c-rel that are similar to splicing signals, indicating that the 5' virus-rel junction of Rev-T may have been formed by cellular RNA splicing machinery. Eight presumed introns have presumably been spliced out of c-rel to generate v-rel. There are also short imperfect regions of homology between sequences at the boundaries of v-rel and sequences in Rev-A and c-rel (turkey), indicating that c-rel may have been transduced by homologous recombination. There are many differences between the amino acid sequences of the predicted translational products of v-rel and c-rel which may account for their difference in transformation potential. These sequence differences between v-rel and c-rel include 10 missense transitions, four missense transversions, and three places where Rev-T has a small in-frame deletion of sequences relative to c-rel. Most of the coding sequence differences between c-rel and v-rel are nonconservative amino acid changes.
Porcine leukocyte cellular subsets sensitive to African swine fever virus in vitro
Authors: Casal I , Enjuanes L , Viñuela E
Published: January 2020

African swine fever virus infected most, if not all, of the macrophages (monocytes) and ca. 4% of the polymorphonuclear leukocytes from porcine peripheral blood. B and T lymphocytes, either resting or stimulated with phytohemagglutinin, lipopolysaccharide, or pokeweed mitogen, were not susceptible to the virus. All of the mitogens used inhibited African swine fever multiplication in susceptible cells. The number of virus passages in vitro and the virulence degree of the virus did not affect the susceptibility of porcine B or T lymphocytes to African swine fever virus.
Specificity of initiation of plus-strand DNA by Rous sarcoma virus
Authors: Smith J K , Cywinski A , Taylor J M
Published: January 2020

We previously reported that in the endogenous reaction of Rous sarcoma virus disrupted by melittin, plus-strand DNA initiates on a small oligonucleotide primer and that this initiation can be reconstructed in vitro in reactions containing purified minus-strand DNA as template, viral RNA as a source of primer, and reverse transcriptase (Smith et al., J. Virol. 49:200-204, 1984). Further studies on the specificity of initiation in the endogenous reaction have shown the following. (i) The primer was 12 nucleotides in length. Its sequence began with a 5' pyrimidine, followed by 11 purines, ending with rGrA-3'. This sequence was in agreement with the known plus-strand RNA sequence immediately upstream from the initiation site. Thus, the primer began one nucleotide 5' to the so-called polypurine tract that has been found on all retrovirus genomes. (ii) The transition point between RNA primer and DNA product was precisely located. It was before the end of the polypurine tract. Thus the polypurine tract, although essential for virus replication and probably a flag for the priming event, did not define the limits of the RNA primer. After primer removal, the DNA had a 5' phosphate, consistent with generation by the viral RNase H activity. The priming specificity in reconstructed reactions was also examined further, with the following observations. (i) When the source of RNA primer was prehybridized to the template viral DNA, the generation, utilization, and subsequent removal of primer were essentially the same as those observed in the endogenous reaction. In the absence of deliberate prehybridization, some specificity was lost. There were than additional locations for the 5' end of the primer as well as the transition point between RNA primer and DNA. (ii) Purine-rich oligoribonucleotides created by RNase A digestion of viral RNA could prime strong-stop plus DNA, but again with the loss of specificity relative to that in the endogenous reaction. (iii) The 5' end of the minus-strand DNA template was not required for initiation of strong-stop plus DNA. Therefore, the specificity of initiation did not depend upon an intramolecular interaction requiring the two inverted repeat sequences that flank the long terminal repeat.
Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53
Authors: Schmieg F I , Simmons D T
Published: January 2020

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.
Transcriptional mapping of human respiratory syncytial virus
Authors: Dickens L E , Collins P L , Wertz G W
Published: January 2020

A transcriptional map for human respiratory syncytial virus was determined by measuring the kinetics of viral gene inactivation in response to UV irradiation. Monolayer cell cultures of respiratory syncytial virus-infected HEp-2 cells were exposed to UV light, and residual viral RNA synthesis was monitored both by gel electrophoresis and by hybridization to dot blots of cloned cDNAs of the 10 known viral genes. Target sizes for the 10 individual viral genes were calculated relative to the UV sensitivity of intracellular viral genome replication. Target size analysis indicated that the 10 viral genes were transcribed as a single transcriptional unit and that the transcription of an individual gene was dependent on the prior transcription of all the preceding genes. The order of gene transcription (with nomenclature according to encoded proteins) was determined to proceed from the promoter as follows: 14K, 11K, N, P, M, 9.5K, 36K, F, 24K, L.
Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA
Authors: White E , Grodzicker T , Stillman B W
Published: January 2020

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.
Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos
Authors: Papkoff J , Ringold G M
Published: January 2020

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.
Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen
Authors: MacArthur H , Walter G
Published: January 2020

Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum.
Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products
Authors: Henderson L E , Sowder R , Copeland T D , Smythers G , Oroszlan S
Published: January 2020

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.
Cellular localization of the transforming protein of wild-type and temperature-sensitive Fujinami sarcoma virus
Authors: Moss P , Radke K , Carter V C , Young J , Gilmore T , Martin G S
Published: January 2020

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.
Lentiviruses are naturally resident in a latent form in long-term ovine fibroblast cultures
Authors: Barban V , Quérat G , Sauze N , Filippi P , Vigne R , Russo P , Vitu C
Published: January 2020

Long-term ovine fibroblast cultures contain replicative-competent lentiviruses in a latent form. This in vitro phenomenon, never described previously for lentiviruses, was clearly demonstrated by activating the expression of latent viruses with various inducing cell treatments, some of which were efficient in inducing endogenous retroviruses or latent herpesviruses. Activated lentiviruses were highly lytic in ovine fibroblasts (type I), or they established persistent infections (type II) as described previously for field isolates from sheep with progressive pneumonia (Quérat et al., J. Virol. 52:671-678, 1984).
Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis
Authors: Gassenmaier A , Lammel M , Pfister H
Published: January 2020

Five human papillomavirus (HPV) DNAs from lesions of an epidermodysplasia verruciformis patient were cloned in lambda L 47: DNA of HPV 5, which predominated in the carcinoma; DNA of a variant type of HPV 8, which was not detected in the carcinoma DNA by Southern blot hybridization but only by cloning; and DNAs of three papillomaviruses that were isolated from warts. Southern blot and liquid phase DNA-DNA hybridization under stringent conditions showed that the three viruses from warts were new types, which we named HPVs 19, 20, and 25. These viruses cross-hybridized between 3 and 29% among themselves and with HPVs 5 and 8. After physical mapping with several restriction enzymes, the colinear genomes were aligned with HPV 8 DNA to define early and late regions. HPVs 8, 19, and 25 shared homology in different parts of their genomes.
Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus
Authors: Vroman B , Luka J , Rodriguez M , Pearson G R
Published: January 2020

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.
Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells
Authors: Smith C , Wolcott J A , Wust C J , Brown A
Published: January 2020

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.
Injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2'-5' oligoadenylate synthetase in peritoneal macrophages
Authors: Gresser I , Vignaux F , Belardelli F , Tovey M G , Maunoury M T
Published: January 2020

Injection of conventional or axenic weanling mice with potent sheep or goat antibody to mouse interferon alpha/beta resulted in a decrease in the basal level of 2-5A synthetase in resting peritoneal macrophages and rendered these cells permissive for vesicular stomatitis virus. There was a good inverse correlation between the level of 2-5A synthetase in peritoneal macrophages and the permissivity of these cells for vesicular stomatitis virus. The peritoneal macrophages of 1- and 2-week-old mice had low levels of 2-5A synthetase and were permissive for vesicular stomatitis virus, whereas at 3 weeks (and after) there was a marked increase in the level of 2-5A synthetase in peritoneal macrophages, and these cells were no longer permissive for vesicular stomatitis virus. We suggest that low levels of interferon alpha or beta or both are produced in normal mice, and that this interferon contributes to host defense by inducing and maintaining an antiviral state in some cells.
Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase
Authors: Sixbey J W , Pagano J S
Published: January 2020

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.
Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein
Authors: Kurath G , Leong J C
Published: January 2020

The genome RNA and six mRNA species of infectious hematopoietic necrosis virus were analyzed by denaturing gel electrophoresis. The following molecular weights were determined: genome RNA, 3.7 X 10(6); mRNA 1, 2.26 X 10(6); mRNA 2, 5.63 X 10(5); mRNA 3, 4.84 X 10(5); mRNA 4 (containing two different mRNA species), 3.00 X 10(5); and mRNA 5, 1.95 X 10(5). Densitometer analyses of gels were used to calculate the molar ratios of the intracellular mRNA species: mRNA 1, 0.02; mRNA 2, 0.49; mRNA 3, 1.0; mRNA 4, 2.52; and mRNA 5, 0.41. Hybrid selection studies determined the mRNA coding assignments as follows: mRNA 1 encodes the viral polymerase, L; mRNA 2 encodes the glycoprotein, G; mRNA 3 encodes the nucleocapsid protein, N; mRNA 4 is composed of two comigrating mRNA species which encode the matrix proteins, M1 and M2; and mRNA 5 encodes a previously unrecognized viral protein which is induced in infected cells but is not present in mature virions. This nonvirion protein has been designated the NV protein.
DNA-binding protein associated with herpes simplex virus DNA polymerase
Authors: Vaughan P J , Purifoy D J , Powell K L
Published: January 2020

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.
Transcriptional activity of avian retroviral long terminal repeats directly correlates with enhancer activity
Authors: Cullen B R , Raymond K , Ju G
Published: January 2020

Retroviral long terminal repeats (LTRs) contain elements responsible for the control of proviral transcription and gene expression. Molecular clones of the LTR region of a number of avian retroviruses have been isolated, and DNA sequence analysis of these clones reveals the existence of a related, but heterogeneous, family of LTRs. To examine the functional significance of the observed sequence differences, we have directly tested the abilities of several different avian retrovirus LTRs to act as promoters and enhancers of mRNA transcription. Our results indicate that large differences in LTR transcriptional activity exist and that these differences in gene expression directly correlate with LTR enhancer activity. In particular, we show that the LTR of Fujinami sarcoma virus is intermediate in both transcriptional and enhancer activity when compared with the very active LTRs of the exogenous viruses RAV-2 and Schmidt-Ruppin B and the much less active LTRs of the endogenous virus RAV-0 and its provirus ev-2. These results suggest that LTR enhancer activity may be the primary determinant of avian retroviral LTR transcriptional activity and, hence, oncogenic potential.
HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells
Authors: Petit M A , Pillot J
Published: January 2020

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.
Characterization of the gene encoding herpes simplex virus type 2 glycoprotein C and comparison with the type 1 counterpart
Authors: Swain M A , Peet R W , Galloway D A
Published: January 2020

The gene encoding the glycoprotein C (gC) of herpes simplex virus type 1 maps to the region of the viral genome from 0.62 to 0.64. Recently, a herpes simplex virus type 2 glycoprotein previously designated gF and now designated gC was mapped to a homologous location. Analysis of the herpes simplex virus type 2 mRNA species encoded in this region revealed a major transcript of 2.5 kilobases, a 0.73-kilobase transcript (the 5' ends of which were mapped by primer extension), and several minor species, all nearly identical to the herpes simplex virus type 1 pattern. A polypeptide of ca. 60,000 daltons was identified by in vitro translation of hybrid-selected mRNA. A smaller protein of ca. 20,000 daltons was also mapped to this region. The nucleotide sequence of a 3.4-kilobase segment of DNA encompassing gC was determined, and an open reading frame of 1,440 nucleotides specifying a 480-amino acid protein with properties consistent with that of a glycoprotein was identified. Comparative DNA sequence analysis showed regions of limited homology within the coding sequences for gC and a deletion which results in 31 fewer amino acids in the gC-2 near the amino terminus of the protein. The carboxy termini of gC-1 and gC-2 are very similar, as are the 20,000-dalton proteins.
Small and middle T antigens contribute to lytic and abortive polyomavirus infection
Authors: Türler H , Salomon C
Published: January 2020

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was less than 10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.