bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of polyomavirus late promoter activity by viral early proteins
Authors: Kern F G , Pellegrini S , Cowie A , Basilico C
Published: January 2020

To assess the effect of the polyomavirus (Py) early proteins, the large T (LT), middle T (MT), and small T (ST) antigens, on gene expression from the Py late promoter, replication-defective plasmid constructs with the bacterial chloramphenicol acetyltransferase (cat) gene linked to this promoter were cotransfected into mouse or rat cells with plasmids capable of producing either LT, MT, or all three early proteins. When target CAT plasmids contained a truncated early region and thus had the coding potential for MT and ST, base-line CAT activities were low, whereas cotransfection with an LT plasmid resulted in up to 70-fold stimulation of CAT activity that was also reflected in similar increases in the level of steady-state mRNA. Studies with target plasmids with deletions within the Py regulatory region indicated that at least the major LT-binding site C and a functional enhancer region were both required for maximal stimulation of CAT activity. However, although enhancer deletions totally suppressed the ability of target plasmids to be trans activated, a consistent two- to fourfold stimulation of CAT activity by LT was still observed with a plasmid in which all three major LT-binding sites were deleted. Of four mutant LTs incapable of binding Py DNA but retaining immortalization potential, only one showed a low but significant trans-activating ability. When the early coding region was completely eliminated from the target plasmid, base-line CAT activity was increased 10-fold. LT failed to stimulate CAT activity to the same levels observed with target plasmid containing the truncated early region, but this limited response could be enhanced by supplying, in addition, MT and ST. Our results suggest that LT trans activation may involve the formation of a complex of transcriptional factors which interacts with the enhancer, an interaction that is facilitated both by the binding of LT to the Py regulatory region and by the presence of MT or ST or both, and that a significant portion of LT stimulation of late gene expression is a result of the removal of the competing early transcriptional unit via autoregulation. In addition, our results suggest that LT trans activation involves a second indirect component acting independently of LT binding and that the immortalization and trans activation functions of LT can be dissociated.
Bovine herpesvirus 1: strain comparison of polypeptides and identification of a neutralization epitope on the 90-kilodalton hemagglutinin
Authors: Trépanier P , Minocha H C , Bastien Y , Nadon F , Séguin C , Lussier G , Trudel M
Published: January 2020

The intracellular and structural polypeptides of the Los Angeles and Cooper 1 reference strains of bovine herpesvirus 1, together with 12 other Canadian field isolates, were analyzed by polyacrylamide gel electrophoresis. Although a few minor differences were noted among some isolates in regard to intracellular viral protein content, analysis of partly purified virus showed strikingly similar polypeptide profiles among 19 proteins with molecular masses of 14 to 145 kilodaltons (kDa). Moreover, a neutralizing monoclonal antibody produced against the Cooper 1 strain also neutralized all of the other 13 strains tested in this study and immunoprecipitated the major 90-kDa glycoprotein. A second monoclonal antibody with a high hemagglutination inhibition titer prevented hemagglutination of other strains tested and also reacted against the 90-kDa glycoprotein by immunoprecipitation, indicating that this glycoprotein is responsible for the hemagglutinating activity of the viral particle and carries an important neutralization epitope.
Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis
Authors: Brightman B K , Pattengale P K , Fan H
Published: January 2020

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).
Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40
Authors: Subramanian S , Bhat R A , Rundell M K , Thimmappaya B
Published: January 2020

Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs.
Detection by monoclonal antibodies of an early membrane protein induced by Epstein-Barr virus
Authors: Balachandran N , Pittari J , Hutt-Fletcher L M
Published: January 2020

Two monoclonal antibodies, E8B3 and E8D2, were raised against Epstein-Barr virus (EBV)-producing cells and were shown to immunoprecipitate a protein with an approximate molecular weight of 105,000 (p105). The protein was detectable only in EBV-containing cells which were supporting the virus lytic cycle, and its synthesis increased after cells were induced with phorbol esters. The molecule was radiolabeled and immunoprecipitated from virus-producing cells that had been extrinsically labeled with 125I, and the antibodies E8B3 and E8D2 reacted in immunofluorescence assays with infected cells; the molecule was also associated with virion particles. Synthesis of p105 was not blocked by phosphonoacetic acid and could be induced in Raji cells by superinfection with virus derived from P3HR1 cells. These data support the conclusion that p105 is an EBV-specific early membrane protein.
Prevention of simian acquired immune deficiency syndrome with a formalin-inactivated type D retrovirus vaccine
Authors: Marx P A , Pedersen N C , Lerche N W , Osborn K G , Lowenstine L J , Lackner A A , Maul D H , Kwang H S , Kluge J D , Zaiss C P
Published: January 2020

Experimental induction of simian acquired immune deficiency syndrome (SAIDS) by inoculation of juvenile rhesus monkeys with a type D retrovirus was prevented by immunization with Formalin-killed whole SAIDS retrovirus serotype 1 containing the adjuvant threonyl muramyl-dipeptide. All six immunized animals developed neutralizing antibody after three injections, while six age-matched cagemates receiving adjuvant alone were antibody free. All 12 monkeys were challenged intravenously with a potentially lethal dose of SAIDS retrovirus serotype 1. The six immunized animals failed to develop persistent viremia and remained clinically normal 8 months postchallenge. In contrast, five of six nonvaccinates developed persistent viremia, four of six developed clinical SAIDS, and two of six died with SAIDS at 10 weeks and 8 months postchallenge, respectively. These results show that prevention of a common spontaneous retrovirus-induced immunosuppressive disease in macaques is now possible by vaccination.
Adsorption of mycoplasmavirus MV-L2 to Acholeplasma laidlawii: effects of changes in the acyl-chain composition of membrane lipids
Authors: Steinick L E , Christiansson A
Published: January 2020

The enveloped mycoplasmavirus MV-L2 and its host Acholeplasma laidlawii JA1 were used to study the ways in which changes in the membrane lipid bilayer affect virus adsorption. The physical state of the membranes was altered by (i) using viruses and bacteria with different membrane lipid acyl-chain compositions, (ii) using incorporation of cholesterol, and (iii) changing the temperature. Adsorption of viruses was strongly dependent on the acyl-chain composition of the virus and the host. Adsorption to homologous hosts was poor, whereas adsorption to hosts with highly different membrane lipid acyl-chain composition was much stronger. We found a heterogeneity within virus populations produced from hosts with different acyl-chain compositions. In a given virus population, various subpopulations differing in acyl-chain composition were found that differed in their ability to adsorb to cells with a specific acyl-chain composition. The adsorption rate increased slightly when cholesterol was present in the viral membranes but decreased considerably when cholesterol was present in the bacterial membranes. The rate of adsorption was temperature dependent with an increase in adsorption rate above 20 degrees C (for hosts with equal amounts of palmitoyl and oleoyl acyl chains). MV-L2 did not adsorb to the persistently L2-infected strain JA1(2R) but adsorbed very well to the virus-resistant strain A(EF22). The physicochemical properties of the lipid matrix of both virus and host are obviously important factors in the adsorption process.
Molecular cloning and analysis of three cDNA clones homologous to human cytomegalovirus RNAs present during late infection
Authors: Martinez J , St Jeor S C
Published: January 2020

Three virus-specific clones were isolated from a cDNA library synthesized from human cytomegalovirus (AD169)-infected cell RNA and cloned into the expression vector lambda gt11. These clones, designated C3, D10, and H10 were each found to express a human cytomegalovirus/beta-galactosidase fusion protein that was reactive with antibody prepared against purified virions. By using the cloned cDNA, we were able to identify the transcripts that code for each gene product and study the kinetics of expression during permissive infection. Our results suggest that at least two of the RNAs undergo posttranscriptional processing and appear in infected cells at immediate-early times. The authentic C3 gene product was identified by probing Western blots with antibody prepared against fusion protein fpC3.
Abelson murine leukemia virus variants with increased oncogenic potential
Authors: Murtagh K , Skladany G , Hoag J , Rosenberg N
Published: January 2020

A number of strains of Abelson murine leukemia virus (A-MuLV) with various abilities to transform cells have been identified. Among these is the A-MuLV-P90 strain, a mutant derived from A-MuLV-P120 that encodes an A-MuLV protein missing sequences that are normally present at the extreme carboxy terminus of P120 (N. Rosenberg and O. N. Witte, J. Virol. 33:340-348, 1980). This virus transforms NIH 3T3 cells efficiently but does not transform a high frequency of lymphoid cells in vitro or in vivo. In this communication, we show that of the relatively few tumors induced by A-MuLV-P90 nearly all contained new variant viruses that stably expressed either larger or smaller A-MuLV proteins. Strains that expressed larger A-MuLV proteins behaved like A-MuLV-P120 in transformation assays, whereas those expressing smaller A-MuLV proteins induced a high frequency of tumors after a short latent period in vivo but failed to transform large numbers of lymphoid cells in vitro. Thus, these latter viruses separated the requirements for in vitro transformation of lymphoid cells from those for tumor induction. All of the variants differed from A-MuLV-P90 in the carboxy-terminal region of the A-MuLV protein, suggesting that sequences in this region play a key role in the ability of the virus to interact with hematopoietic cells in vivo and in vitro.
The promoter for the late gene encoding Vp5 of herpes simplex virus type 1 is recognized by cell extracts derived from uninfected cells
Authors: Chisholm G E , Summers W C
Published: January 2020

The ability of whole-cell extracts from uninfected HeLa cells to recognize the promoter for the herpes simplex virus type 1 late gene encoding the major capsid protein Vp5 was investigated by using both in vitro transcriptional and S1 nuclease protection analysis. This gene promoter was recognized by the cell extracts and produced abundant amounts of transcript in the absence of any other virus-encoded factors. This transcript was shown to arise, in vitro, from specific initiation at or very near the physiological mRNA start site. Thus, it appears that cell extracts from uninfected HeLa cells can efficiently recognize both early- and late-gene promoters.
Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI
Authors: Larsson S , Svensson C , Akusjärvi G
Published: January 2020

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.
Multiple transforming regions of human cytomegalovirus DNA
Authors: el-Beik T , Razzaque A , Jariwalla R , Cihlar R L , Rosenthal L J
Published: January 2020

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.
Pseudotyped retroviral vectors reveal restrictions to reticuloendotheliosis virus replication in rat cells
Authors: Embretson J E , Temin H M
Published: January 2020

Reticuloendotheliosis viruses (Rev) replicate in chicken and dog cells, but not in rat cells. Amphotropic murine leukemia viruses (Am-MLV) replicate in chicken, dog, and rat cells. Transcription from the Rev long terminal repeat, determined by the chloramphenicol acetyltransferase assay, was not significantly different from transcription from the MLV long terminal repeat in rat cells. To determine further the step(s) in the retroviral life cycle that is blocked for Rev replication in rat cells, we took advantage of the wide host range of Am-MLV (S. Rasheed, M. B. Gardner, and E. Chan, J. Virol. 19:13-18, 1976) and the ability to form Rev-Am-MLV pseudotypes. Data from these pseudotypes indicate that the block to Rev replication in rat cells is posttranscriptional.
N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells
Authors: Puddington L , Bevan M J , Rose J K , Lefrançois L
Published: January 2020

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.
fim-1 and fim-2: two new integration regions of Friend murine leukemia virus in myeloblastic leukemias
Authors: Sola B , Fichelson S , Bordereaux D , Tambourin P E , Gisselbrecht S
Published: January 2020

The Friend helper murine leukemia virus (F-MuLV) induces in mice a high percentage of myeloblastic leukemias. Myeloblastic transformation is also observed after in vitro infection of long-term bone marrow cultures. To investigate the molecular events leading to the generation of myeloblastic leukemias, we first screened a panel of leukemic cells for rearrangement or amplification of known oncogenes or previously described specific integration sites. No modification of these genes was observed. Therefore, we searched for common integration sites by constructing a genomic library from a myeloblastic cell line harboring only five integrated proviruses. This library was screened with a virus-specific probe, and virus-host cellular junction fragments were subcloned. Two flanking cellular sequences corresponding to two different integrated proviruses were used to analyze additional myeloblastic leukemias. The first probe detected rearrangements in 2 of 42 myeloblastic leukemias, and the second probe detected rearrangements in 6 of 42. We demonstrated that, in each case, the rearrangement was the result of F-MuLV integration, with all proviruses in the same orientation and clustering in a region less than 3 kilobases long. The two regions, named fim-1 and fim-2, were different from 15 oncogenes tested. Rearrangements of these two regions were found in F-MuLV-induced myeloblastic leukemias but not in 20 lymphoid or erythroid leukemias induced by the same virus.
Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts
Authors: Berger H , Wintersberger E
Published: January 2020

Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild-type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication.
Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins
Authors: Stein R B , Tai J Y , Scolnick E M
Published: January 2020

Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.
Selective shedding and congenital transmission of endogenous avian leukosis viruses
Authors: Smith E J , Salter D W , Silva R F , Crittenden L B
Published: January 2020

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.
Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus
Authors: Resnick J , Shenk T
Published: January 2020

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.
Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2
Authors: Cohen E A , Gaudreau P , Brazeau P , Langelier Y
Published: January 2020

Herpes simplex virus type 1 ribonucleotide reductase is associated with two polypeptides of apparent molecular weights 136,000 and 38,000. The two polypeptides form a tight complex and, therefore, are often coprecipitated by monoclonal antibodies. We report here that immunoglobulins G purified from polyclonal rabbit antisera (P9) raised against a nonapeptide corresponding to the carboxy terminus of the 38,000-dalton polypeptide specifically neutralize the herpes simplex virus ribonucleotide reductase activity. We suggest that the P9 immunoglobulin G neutralizes the reductase activity by impairing the association of the two subunits (H1 and H2) of the enzyme.
The bacteriophage T4 regulatory protein gpunf/alc binds to DNA in the absence of RNA polymerase
Authors: Snustad D P , Haas N , Oppenheimer D G
Published: January 2020

DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf/alc. The unf/alc protein does not bind to DNA via an association with RNA polymerase; gpunf/alc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography.
Sequence of the genome ends and of the junction between the ends in concatemeric DNA of pseudorabies virus
Authors: Harper L , Demarchi J , Ben-Porat T
Published: January 2020

The nucleotide sequences of the termini of the mature pseudorabies virus genome and of the junction between these termini in concatemeric DNA were compared. To ensure conservation of unmodified 5' and 3' termini, the end fragments obtained directly (uncloned) from mature viral DNA were sequenced. The sequence obtained from 5' and 3' end labeling revealed that whereas the L terminus was blunt ended, the S terminus had a 2-base (GG) 3' overhang. The sequences spanning the junction between the termini present in concatemeric DNA was also determined and compared with that expected when the two ends of the mature DNA were juxtaposed. This comparison showed that in concatemeric DNA the ends of the mature genome had become joined by blunt-end ligation of one of the strands and that the 2-nucleotide gap on the other strand had been repaired. A significant degree of homology between the sequences spanning the junction between the ends of the varicella-zoster virus and pseudorabies virus genomes was found.
In vivo transcription of bacteriophage phi 29 DNA: transcription initiation sites
Authors: Barthelemy I , Salas M , Mellado R P
Published: January 2020

The initiation sites of the RNA transcripts synthesized in vivo in Bacillus subtilis infected with bacteriophage phi 29 have been mapped by S1 protection experiments. Nine transcription initiation sites were localized along the entire phi 29 genome, close to previously reported B. subtilis and Escherichia coli RNA polymerase-binding sites. Eight of these sites corresponded to early transcription and only one corresponded to late transcription. By using 5'-end-labeled RNA, four of the early sites and the late one were shown to be the main sites where initiation of transcription occurs in vivo in the phi 29 genome.
Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen
Authors: Cote P J , Shapiro M , Engle R E , Popper H , Purcell R H , Gerin J L
Published: January 2020

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.
Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene
Authors: Heilbronn R , Jahn G , Bürkle A , Freese U K , Fleckenstein B , zur Hausen H
Published: January 2020

The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.
Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene
Authors: Kouzarides T , Bankier A T , Satchwell S C , Weston K , Tomlinson P , Barrell B G
Published: January 2020

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. We present here a 5280-base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, we were able to analyze the extent of homology between the DNA polymerases of three distantly related herpesviruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpesviruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type 1 and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.
Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site
Authors: Stoltzfus C M , Lorenzen S K , Berberich S L
Published: January 2020

Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed.
Successful infection of the common marmoset (Callithrix jacchus) with human varicella-zoster virus
Authors: Provost P J , Keller P M , Banker F S , Keech B J , Klein H J , Lowe R S , Morton D H , Phelps A H , McAleer W J , Ellis R W
Published: January 2020

The common marmoset, Callithrix jacchus, can be infected with human varicella-zoster virus (VZV), both wild-type strain KMcC and attenuated vaccine strain Oka/Merck. Infection was accomplished with either whole-cell-associated or cell extract VZV by combined oral-nasal-conjunctival application and was characterized by substantial and persistent anti-VZV antibody responses. The infectivity of VZV for marmosets was destroyed by treatment of inocula with heat or UV light. Diluted inocula with as few as 40 PFU/ml were infectious for marmosets. The lungs were demonstrated to be a major site of viral replication; both the presence of viral antigens and signs of pneumonia were demonstrated in lung tissues. Four serial passages of VZV KMcC were carried out in C. jacchus by a process of in vitro isolation and culturing of VZV from infected lung tissue and reapplication of the cultured isolates to fresh animals. The isolated viruses were identified as VZV both serologically and by restriction endonuclease analyses. The C. jacchus infectivity model should prove useful for determining the efficacy of subunit and live recombinant VZV vaccines as well as for the study of zoster.
Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants
Authors: Farkas D H , Block T M , Hart P B , Hughes R G
Published: January 2020

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.
Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californica nuclear polyhedrosis virus genome
Authors: Oellig C , Happ B , Müller T , Doerfler W
Published: January 2020

In several parts of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, nested sets of overlapping RNAs with common 3' or 5' termini have been recognized. In the present report, the pattern of viral transcription and the arrangement of viral gene products in the region of 81.2 to 85.0 map units were investigated. In this segment of the AcNPV genome, at least nine size classes of viral RNA were identified which ranged in size from 1.3 kilobases (kb) to 4.6 kb and exhibited common 3' termini. The detailed restriction map and the nucleotide sequence of this part of the AcNPV genome were determined. Computer analyses revealed several open reading frames (ORFs) on the rightward-transcribed strand with potential TATA and CAAT signals preceding many of the potential ORFs and the 5' termini of some of the mapped RNAs. The leftward-transcribed strand was devoid of major ORFs. The presumptive polypeptides encoded by the larger ORFs ranged in size from 11.3 to 55.6 kilodaltons (kDa). The amino acid sequence of the presumptive polypeptide encoded by ORF3, a 33.6-kDa molecule, exhibited an unusual, clustered 16-fold repeat of the dipeptide arginine-serine in a protein that showed an overall preponderance of basic amino acids. The results of in vitro translation experiments with hybrid-selected RNAs homologous to internal subfragments of the 81.2- to 85.0-map-unit region yielded polypeptides of approximately 28, 34 to 36, and 48 to 50 kDa, which were close in size to the lengths of the major ORFs derived from the nucleotide sequence. The localizations of individual size classes of RNAs in the 81.2- to 85.0-map-unit region of the viral genome were determined precisely at the 3' and 5' termini by S1 protection analyses. Within a sequence of eight nucleotides, all RNAs had the same 3' terminus, which lay close to multiple polyadenylation signals. The initiation sites of the nine different RNA size classes were precisely mapped. As the cap sites of the smaller RNAs (less than 1.8 kb) were determined by S1 protection analyses, a multitude of RNA initiation sites became apparent. It was also shown that the different RNA size classes in the 81.2- to 85.0-map-unit region were detectable as early as 2 h and at least until 36 to 48 h after infection. In unselected cytoplasmic RNA, the size classes of viral RNAs specific for the EcoRI J fragment were detectable early as well as late after infection, although at early times the larger RNAs were detectable in smaller amounts.(ABSTRACT TRUNCATED AT 400 WORDS)
Isolation and characterization of temperature-sensitive mutants of adenovirus type 7
Authors: Praszkier J , Ginsberg H S
Published: January 2020

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.
A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion
Authors: Somasundaran M , Robinson H L
Published: January 2020

In vitro studies indicate that human immunodeficiency virus (HIV) infections are cytopathic for T4+ peripheral blood lymphocytes and for most continuous lines of T4+ lymphocytes. These cytopathic effects have been largely attributed to the formation of syncytia by HIV-infected cells. We report that HIV infections killed cultured peripheral blood lymphocytes and a line of T4+-lymphoid cells (CEM cells) without causing cell fusion. We also report that the occurrence of syncytia is an early and transitory phenomenon following infection of a fusion-susceptible line of T4+-cells (H9 cells). Mixing experiments and flow cytometry have been used to demonstrate that susceptibility to HIV-induced fusion is not determined by differences in presentation of viral envelope antigens or the surface levels of T4 receptor antigens on fusion-susceptible and -resistant cells. We conclude that a major mechanism of HIV-induced cell killing does not involve cell fusion and that HIV-induced cell fusion, when it does occur, requires factors in addition to viral envelope antigens and host T4 receptors.
Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication
Authors: Evans E , Traktman P
Published: January 2020

We have identified a gene encoded by vaccinia virus which is essential for DNA replication. The gene, located in the HindIII D fragment of the viral genome, is transcribed early after infection into two transcripts of 3.0 and 3.7 kilobases which share a 3' terminus. The lesions of three temperature-sensitive DNA replication mutants with defects in this gene have been localized by marker rescue with progressively smaller DNA fragments. We have determined by hybrid selection that the gene encodes an 82-kilodalton protein. An antibody has been prepared against this polypeptide and used to quantitate expression of the protein after infection with wild-type virus or with a viral mutant whose lesion maps within this gene. The temporal pattern of expression in the mutant is unaffected, but the product encoded by the mutant is significantly more thermolabile than the wild-type protein.