bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific interaction of simian virus 40 large T antigen with cellular chromatin and nuclear matrix during the course of infection
Authors: Schirmbeck R , Deppert W
Published: January 2020

We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.
Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells
Authors: Patick A K , Hinze H C
Published: January 2020

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.
Polyomavirus early region alternative poly(A) site: 3'-end heterogeneity and altered splicing pattern
Authors: Norbury C J , Fried M
Published: January 2020

The position of an alternative polyadenylation [poly(A)] site at the 3' end of the polyomavirus middle T antigen (T-Ag) coding sequences suggests the possibility of a functional role for this site in early gene regulation. The fine structure of this alternative poly(A) site was determined by cDNA sequence and 3' S1 analyses. Cleavage-poly(A) was found to be heterogeneous, occurring at multiple CA dinucleotides downstream from the AATAAA signal sequence. About 50% of the alternative poly(A) takes place upstream from the middle T-Ag stop codon. In addition, the pattern of splicing of transcripts with the alternative poly(A) site differed from that with the major poly(A) site at the end of the early region. The ratio of the small and middle T-Ag splices to the large T-Ag splice for the alternative poly(A)+ mRNAs is about 2.5 times that found for mRNAs with the major poly(A) site. The altered splicing pattern and 3'-end heterogeneity of the alternative poly(A)+ mRNAs would result in preferential translation of small T-Ag (to a greater degree) and middle T-Ag over large T-Ag at later times in the polyomavirus lytic cycle.
Wild-type bacteriophage T4 is restricted by the lambda rex genes
Authors: Shinedling S , Parma D , Gold L
Published: January 2020

The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens. The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well. Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex.
Mapping bovine herpesvirus type 1 latency-related RNA in trigeminal ganglia of latently infected rabbits
Authors: Rock D L , Beam S L , Mayfield J E
Published: January 2020

Here we have used the bovine herpesvirus type 1 (BHV-1) rabbit model together with in situ nucleic acid hybridization to identify and map viral RNA present in latently infected neurons. Radioactively labeled cloned HindIII fragments representing most of the BHV-1 genome (Cooper strain) were individually hybridized to sections of trigeminal ganglia taken from rabbits during acute and latent stages of infection. Whereas all viral genomic fragments hybridized to lytically infected tissue culture cells and to acutely infected ganglia, only HindIII fragment D (map units 0.734 to 0.842) hybridized to latently infected ganglionic neurons. Additional in situ hybridization experiments using subcloned fragments of HindIII-D further mapped the latency-related viral RNA to a 1.9-kilobase region (map units 0.734 to 0.748) of the viral genome. These results indicate that BHV-1 gene transcription is restricted during the latent phase of infection; further, they suggest that specific viral transcription may be involved in establishment or maintenance of latent BHV-1 infection.
Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells
Authors: Jamieson B D , Butler L D , Ahmed R
Published: January 2020

The lifelong chronic lymphocytic choriomeningitis virus (LCMV) infection established in neonatally or congenitally infected mice can be eliminated by adoptive transfer of lymphoid cells from LCMV-immune mice. In this study, we have identified the effector cells mediating the clearance of persistent and disseminated LCMV infection. Using mice that are recombinant in the H-2 region and by selective depletion of lymphocyte subpopulations, we show that viral clearance was mediated by LCMV-specific Lyt2+ L3T4- T cells that are restricted to the class I genes of the major histocompatibility complex. In addition, our results show a requirement for host-derived bone marrow cells for the effective elimination of virus from the liver. These studies emphasize the importance of virus-specific T cells and an intact bone marrow function in viral clearance.
A small deletion distant from a splice or polyadenylation site dramatically alters pre-mRNA processing in region E3 of adenovirus
Authors: Bhat B M , Wold W S
Published: January 2020

The E3 complex transcription unit of adenovirus encodes overlapping mRNAs (a to i) with different exon structures. The major mRNAs are a (approximately 40% of the total) and c (approximately 15%), which are spliced once, and f (approximately 15%) and h (approximately 25%), which are spliced twice. mRNA a uses the upstream E3A polyadenylation site, and the other mRNAs use the downstream E3B polyadenylation site. We analyzed virus deletion mutants to identify sequences important in alternative pre-mRNA processing in region E3. Our main finding is that a 64-base-pair deletion in dl742 causes mainly mRNAs f and h to be formed. mRNAs a and c are barely made. dl742 does not delete either a splice site or a polyadenylation site. Thus, the sequences deleted must function in alternative pre-mRNA processing independently of the signals at the actual splice and polyadenylation sites. The lack of synthesis of mRNA a by dl742 does not appear to result from a defect in the E3A polyadenylation signal but rather from an increase in splicing activity which results in the synthesis of doubly spliced mRNAs f and h at the expense of singly spliced mRNAs a and c. This suggests, in the wild-type situation, that the frequency of use of the E3A versus the E3B polyadenylation site may be determined by the rate of splicing, as well as, presumably, the rate of cleavage-polyadenylation at the E3A site.
A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin
Authors: Tsurumi T , Maeno K , Nishiyama Y
Published: January 2020

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.
Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain
Authors: Alexander F , Leis J , Soltis D A , Crowl R M , Danho W , Poonian M S , Pan Y C , Skalka A M
Published: January 2020

Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.
Differential effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine on herpes simplex virus and Epstein-Barr virus in a dually infected human lymphoblastoid cell line
Authors: van der Horst C M , Lin J C , Raab-Traub N , Smith M C , Pagano J S
Published: January 2020

We investigated the effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on a lymphoblastoid cell line dually infected with Epstein-Barr virus and herpes simplex virus (HSV) type 1. The numbers of Epstein-Barr virus genomes were reduced during 70 days of treatment with either drug. Both drugs suppressed HSV replication in a dose-related manner. In the continued presence of the drugs, HSV developed resistance, rapidly to acyclovir and much more slowly to 30 microM DHPG. Analysis of HSV glycoprotein C production and viral DNA showed that treatment with 100 microM DHPG eliminated HSV production, curing the cell line of HSV persistent infection.
Expression of the art gene protein of human T-lymphotropic virus type III (HTLV-III/LAV) in bacteria
Authors: Goh W C , Sodroski J G , Rosen C A , Haseltine W A
Published: January 2020

Human T-lymphotropic virus type III (HTLV-III/LAV or HIV) contains a gene designated art (anti-repressor transactivator). Here, we report the expression of the art gene product in bacteria and show that the 20-kilodalton (kDa) bacterially expressed art protein is recognized by serum of a patient. The bacterially synthesized art protein competed in an immunological reaction with a 20-kDa protein produced in HTLV-III/LAV-infected lymphocytes. Antiserum to a synthetic oligopeptide corresponding to a sequence in the second exon of the art gene also precipitated the 20-kDa protein in HTLV-III/LAV-infected cells. These results demonstrate that the 20-kDa art gene product is expressed in cell lines that produce HTLV-III/LAV virions.
Inhibition of vesicular stomatitis viral mRNA synthesis by interferons
Authors: Belkowski L S , Sen G C
Published: January 2020

Interferon (IFN) treatment inhibited the replication of vesicular stomatitis virus (VSV) in human GM2767 and mouse JLSV-11 cells. The replication of this virus in either human RD-114 or mouse A402 cells was insensitive to IFN treatment. We analyzed various steps in the VSV life cycle as they occurred under different conditions of IFN treatment to identify the point(s) at which IFN was exerting its inhibitory effect. IFN treatment led to strong inhibition of viral protein synthesis and accumulation of viral RNA in both lines of IFN-sensitive cells. No such effect was observed in the IFN-resistant cells. Using a temperature-sensitive mutant (tsG41) and wild-type VSV that were not undergoing protein synthesis, we determined that the major site of action of IFN against VSV replication in JLSV-11 and GM2767 cells was at the level of primary viral transcription. The accumulation of primary viral transcripts was strongly inhibited in these cells by IFN treatment. This effect was not a consequence of any effect of IFN on virus entry and uncoating. Thus, it appears that IFN exerts a direct effect on the VSV transcriptional process in GM2767 and JLSV-11 cells.
Simian virus 40 associates with nuclear superstructures at early times of infection
Authors: Watson J B , Gralla J D
Published: January 2020

The association of infecting simian virus 40 with insoluble nuclear structures was assayed by disrupting infected nuclei and assaying insoluble fractions for virus. Three methods were used which lyse nuclei but maintain the insolubility of residual nuclear structures: sonication, high-salt-Triton-EDTA extraction, and low-salt-lithium diiodosalicylate extraction. After each type of nuclear extraction, infecting simian virus 40 remained associated with the residual nuclear structures. This association depended strictly on natural viral infections and on the use of buffers containing moderate amounts of salt and Mg2+ for the isolation of infected nuclei. These viral interactions exhibited behavior similar to host cell DNA interactions studied by analogous assays. Both viral DNA and coat proteins were found associated with the host cell nuclear superstructure. We concluding that at early times after infection the viral templates mimic the state of the host cell chromatin by attaching to the cellular nuclear matrix.
Host factor-induced template modification during synthesis of poliovirus RNA in vitro
Authors: Hey T D , Richards O C , Ehrenfeld E
Published: January 2020

Poliovirus RNA polymerase, 3Dpol, transcribes poliovirus RNA in vitro in the presence of a host factor (HF) preparation from uninfected HeLa cells to yield heterogeneous-size product RNAs. The products include some molecules larger than the template that represent self-primed elongations of the template from a 3'-terminal hairpin. We showed that transcription proceeded through the formation of a modified RNA intermediate that was generated by nucleolytic cleavage of the template by HF in the absence of nucleoside triphosphates. Cleavage resulted in the loss of the original poly(A) 3' end and the generation of new, heterogeneous 3' ends that formed self-priming structures that could then be elongated by 3Dpol or reverse transcriptase. The two stages of the reaction, (i) cleavage to yield self-priming templates and (ii) subsequent chain elongation to yield heterogeneous-size products up to nearly dimer length, could be separated. RNAs whose original 3' ends were chemically oxidized so as to prevent chain elongation showed no reduction in template activity after preincubation with HF. We conclude that an HF preparation that contains a low level of nuclease activity is sufficient to activate RNA templates for transcription by 3Dpol to generate up to apparent dimer-length products. This reaction likely has little relevance to the mechanism of poliovirus RNA replication in vivo. It is likely that numerous other factors or activities, in addition to 3Dpol, would also result in transcription of poliovirus RNA in vitro.
Inhibition of Cleavage of a Plant Viral Polyprotein by an Inhibitor Activity Present in Wheat Germ and Cowpea Embryos
Authors: Shih Ding S. , Bu Ming , Price Mary A. , Shih Chao-Yun T.
Published: January 2020

In rabbit reticulocyte lysate, the bottom component RNA of cowpea mosaic virus directs the synthesis of a 200,000-molecular-weight precursor protein (200K protein) that is cleaved during synthesis by a reticulocyte enzyme to form a 32K protein and a 170K protein. Cleavage of the 200K protein was found to be effectively inhibited by inhibitor activity in wheat germ and cowpea embryo extracts. The inhibitor was nondialyzable, precipitatable by ammonium sulfate, and partially stable at high temperatures. The activity appeared to be specific in that it caused no inhibition of the secondary cleavage reactions (cleavage of the 170K protein) at concentrations that were sufficient to cause complete inhibition of the primary cleavage reaction (cleavage of the 200K protein).
Role of the agnoprotein in regulation of simian virus 40 replication and maturation pathways
Authors: Hou-Jong M H , Larsen S H , Roman A
Published: January 2020

Analysis of two agnogene mutants, dl2304 deleted over the entire agnogene and in2379 carrying a 2-base insert, indicated that the mutant phenotype of small plaque formation must be the result of a defect late in the maturation pathway. Both mutants were removed from the pool of molecules available for replication with wild-type kinetics. Whereas dl2304 was somewhat reduced in its rate of progression from chromatin to previrions-virions, in2379, which produced even smaller plaques than dl2304 did, progressed with wild-type kinetics. Therefore, the agnoprotein was not required for progression from chromatin to previrions.
Dimers and complexes with p53 are the prevalent oligomeric forms of a transforming nonkaryophilic T antigen of simian virus 40
Authors: Montenarh M , Vesco C , Scheidtmann K H
Published: January 2020

The oligomers formed by a mutant nonkaryophilic large T antigen of simian virus 40, which lacks residues 110 through 152 of normal large T antigen and transforms only established cells (L. Fischer-Fantuzzi and C. Vesco, Proc. Natl. Acad. Sci. USA 82:1891-1895, 1985), were found to consist predominantly of dimers. Anti-p53 antibodies precipitated 14 to 16S complexes containing the mutant nonkaryophilic large T antigen and p53 from extracts of transformed cells, and anti-p53 indirect immunofluorescence stained these cells in the cytoplasm.
Horizontal transmission of murine retroviruses
Authors: Portis J L , McAtee F J , Hayes S F
Published: January 2020

Both a feral mouse ecotropic virus (WM-E) and Friend ecotropic virus (F-MuLV) were transmitted horizontally among adult mice. Infection resulted in the production of antiviral antibody in the recipients, with no evidence of viremia or clinical disease. However, persistent low-level virus replication was detectable in the spleens of these mice as long as 8 months after initial infection. External secretions, including saliva, semen, and uterine secretions from viremic mice contained high concentrations of infectious virus. Nevertheless, transmission occurred only from viremic males to either males or females. Male-to-male transmission appeared to occur by parenteral inoculation of infectious saliva during fighting behavior. Evidence is presented that infection of females was by the venereal route. Of four mouse strains examined, NFS/N, IRW, and C57L females were all susceptible to venereal infection, whereas AKR mice were not. Since AKR mice are susceptible to infection by WM-E administered parenterally, this resistance appeared to be mediated by local viral interference due to the high-level expression of endogenous Akv gp70 within the female reproductive tract. Although both WM-E and F-MuLV were transmitted from viremic males to females, infection by WM-E was significantly more efficient than that by F-MuLV. This difference correlated with a distinct difference in cellular tropism of WM-E and F-MuLV within the epididymis of viremic males. F-MuLV gp70 was expressed only within stromal elements, whereas WM-E gp70 was seen largely within the epithelial lining cells and luminal contents of the duct. No evidence of virus expression within germ cells was observed. The possible influence of virus expression by epithelial cells of the female reproductive tract on infection of embryos is discussed.
Anti-influenza virus cytotoxic T lymphocytes recognize the three viral polymerases and a nonstructural protein: responsiveness to individual viral antigens is major histocompatibility complex controlled
Authors: Bennink J R , Yewdell J W , Smith G L , Moss B
Published: January 2020

It has recently been shown that antiviral major histocompatibility complex class I-restricted cytotoxic T lymphocytes can recognize proteins that serve as internal viral structural components (influenza A virus nucleoprotein, vesicular stomatitis virus nucleocapsid protein). To further examine the role of internal viral proteins in cytotoxic T-lymphocyte recognition, we constructed recombinant vaccinia viruses containing individual influenza A virus genes encoding three viral polymerases (PB1, PB2, PA) and a protein not incorporated into virions (NS1). We found that cells infected with each of these recombinant vaccinia viruses could be lysed by anti-influenza cytotoxic T lymphocytes. Cytotoxic T-lymphocyte responsiveness to the individual viral antigens varied greatly between mouse strains. By using congenic mouse strains, responsiveness to PB1 and PB2 was found to cosegregate with major histocompatibility complex haplotype. These findings provide further evidence that internal antigens play a critical role in cytotoxic T-lymphocyte recognition of virus-infected cells. Additionally, they suggest that the cytotoxic T-lymphocyte response to viral antigens may often be restricted to only a fraction of the major histocompatibility complex class I repertoire.
Characterization of Rous sarcoma virus sequences essential for viral gene expression
Authors: Norton P A , Coffin J M
Published: January 2020

Using the Escherichia coli lacZ gene product beta-galactosidase as an indicator of gene expression, we analyzed sequences that are required for expression of the Rous sarcoma virus (RSV) genome in avian cells. The RSV long terminal repeat (LTR) and leader region were sufficient to direct the synthesis of high levels of enzymatically active gag-lacZ fusion proteins. A portion of U3 greater than 140 nucleotides upstream from the cap site was essential for gene expression. This element functioned in either orientation, but its activity was attenuated when it was relocated further away from the cap site. The insertion of exogenous LTRs 3' of lacZ augmented the expression of that gene by increasing the level of stable gag-lacZ transcripts. Furthermore, 3' LTRs could partially compensate for certain defects within the 5' LTR. Insertion of various fragmentary LTRs allowed the identification of at least three synergistically acting domains within the 3' LTR that influence gene expression. Interestingly, the gag-lacZ expression was only stimulated by a 3' LTR when the exogenous 3'-untranslated region was adjacent. Our results imply that the two LTRs of a provirus interact in a complex manner to promote high levels of stable transcripts. It was also found that gag-lacZ expression was independent of viral gene products, suggesting that trans-activation is not a key mechanism regulating RSV expression in avian cells.
Transition from a heterozygous to a homozygous state of a pair of loci in the inverted repeat sequences of the L component of the herpes simplex virus type 1 genome
Authors: Umene K
Published: January 2020

The behavior of herpes simplex virus type 1 heterozygous isolates, in which the two inverted repeats of the L component (RL) were differentiated by a polymorphism marker (the presence [type B] or absence [type A] of a SalI site), was investigated. The progeny viruses derived from the heterozygote (A/B) consisted of heterozygotes (A/B), type A homozygotes (A/A), and type B homozygotes (B/B). The heterology between RL, albeit tolerated, was unstable, as is the case with heterology between the repeats of the S component. The two repeats TRL (terminal) and IRL (internal) were equipotent in generating homozygotes from a heterozygote. Data obtained from an analysis of 426 progeny viruses derived from heterozygous clones supported the hypothesis that the two loci in RL of a herpes simplex virus type 1 genome are determined as a random combination of the corresponding two loci in RL of the parent virus and that the ratio of heterozygotes/type A homozygotes/type B homozygotes in the progeny viruses from a heterozygote is expected to be 2:1:1. An ephemeral dominance of one type of homozygote over the other was observed in subclones from several heterozygous clones.
Characterization of middle T antigen expressed by using an adenovirus expression system
Authors: Schaffhausen B S , Bockus B J , Berkner K L , Kaplan D , Roberts T M
Published: January 2020

The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60c-src. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60c-src remained unassociated. Immunoaffinity purification resulted in approximately 100 micrograms of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74- and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.
The 3' long terminal repeat of a transcribed yet defective endogenous retroviral sequence is a competent promoter of transcription
Authors: Levy D E , McKinnon R D , Brolaski M N , Gautsch J W , Wilson M C
Published: January 2020

Although actively transcribed and present as multiple genomic copies, a distinct class of endogenous murine leukemia virus-related sequence does not give rise to infectious virus. Since the long terminal repeat at the 3' terminus provides the transcriptional start site after reintegration, we determined the structure and potential promoter activity of that sequence obtained from cDNA of endogenous retroviral transcripts. These studies demonstrate that the distinctive 3' long terminal repeat sequence of these transcripts could serve as an effective promoter of transcription and, therefore, may not be the primary defect in the infectious cycle of retroviral replication but may result in the propagation of these endogenous retroviral sequences in the genome as retrotransposons.
Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha
Authors: Upton C , Macen J L , McFadden G
Published: January 2020

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.
Site-specific antibodies define a cleavage site conserved among arenavirus GP-C glycoproteins
Authors: Buchmeier M J , Southern P J , Parekh B S , Wooddell M K , Oldstone M B
Published: January 2020

Arenaviruses share a common strategy for glycoprotein synthesis and processing in which a mannose-rich precursor glycoprotein, termed GP-C in lymphocytic choriomeningitis virus (LCMV), is posttranslationally processed by oligosaccharide trimming and proteolytic cleavage to yield two structural glycoproteins, GP-1 and GP-2. Mapping the orientation and proteolytic cleavage site(s) in such polyproteins has traditionally required direct protein sequencing of one or more of the cleaved products. This technique requires rigorous purification of the products for sequencing and may be complicated by amino-terminal modifications which interfere with sequence analysis. We used an alternative approach in which synthetic peptides corresponding to sequences bracketing a potential protease cleavage site were used to raise antisera which define the boundaries of the cleaved products. We found that cleavage of LCMV GP-C to yield GP-1 and GP-2 occurs within a 9-amino-acid stretch of GP-C which contains a paired basic amino acid group -Arg-Arg-, corresponding to amino acids 262 to 263 in the LCMV GP-C sequence. By comparison with the predicted amino acid sequences of a second LCMV strain, LCMV-WE, as well as with the deduced amino acid sequences of the New World arenavirus Pichinde and the Old World virus Lassa, we observed similar conservation of paired basic and flanking amino acid sequences among these viruses.
Characterization and nucleotide sequence of two herpes simplex virus 1 genes whose products modulate alpha-trans-inducing factor-dependent activation of alpha genes
Authors: McKnight J L , Pellett P E , Jenkins F J , Roizman B
Published: January 2020

Herpes simplex viruses encode a structural protein which induces, in trans, expression of alpha genes, the first set of genes to be expressed after infection of permissive cells. This protein, designated as the alpha-trans-inducing factor (alpha-TIF), maps within the BamHI F fragment, and its gene has been sequenced. In the course of mapping the domain of the alpha-TIF gene, it was noted that the intact BamHI fragment was consistently more effective than the complete domain of the alpha-TIF gene in inducing expression of alpha genes. Cotransfections of DNA fragments containing an alpha indicator gene and the alpha-TIF gene with various regions of the BamHI F DNA fragment revealed that the sequences located 3' to the alpha-TIF gene raised the activity of the alpha-TIF gene to nearly the same level as that of the intact BamHI F fragment. The nucleotide sequence and S1 nuclease mapping analyses revealed the presence of two transcribed open reading frames capable of encoding polypeptides with translated molecular weights of 77,357 and 70,527. To determine whether the effect of these sequences in trans on alpha-TIF-mediated induction of alpha genes was due to expression of these genes or competition for transcriptional factors, we constructed plasmids that contained both genes. Into each or both of these genes we inserted, near the translation initiation sites, 14-base-pair linkers carrying translational stop codons (TAG) in all three reading frames. Analyses of these plasmids indicated that the gene encoding the 70,527-molecular-weight polypeptide reduced alpha-TIF-dependent induction of alpha genes, whereas the gene encoding the 77,357-molecular-weight polypeptide increased this activity. Insertion of the stop codons abolished these activities.
Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells
Authors: Eisenlohr L C , Gerhard W , Hackett C J
Published: January 2020

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.
Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step
Authors: Dezélée S , Blondel D , Wyers F , Petitjean A M
Published: January 2020

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.
Productive infection of cultured human lymphoid cells by adenovirus
Authors: Lavery D , Fu S M , Lufkin T , Chen-Kiang S
Published: January 2020

We investigated infection of cultures from established human B- and T-cell lines by adenoviruses. Infection by adenovirus type 2 or 5 was productive by the criteria of viral DNA replication, RNA synthesis, immunofluorescent staining of viral proteins, and assembly of biologically active virions. Whereas the kinetics of infection were reproducible and characteristic for each cell line, there appeared to be no correlation between the kinetics of infection and the origin from which the cell lines were established. In a myeloma and a T-cell line, the kinetics of infection approached those in HeLa cells. The presence of the Epstein-Barr virus genome in B lymphoid cells was not a prerequisite for adenoviral infection. Furthermore, expression of the E1A gene was repressed in myeloma cells in comparison with HeLa cells.
Structural characteristics of the Corynebacterium lilium bacteriophage CL31
Authors: Trautwetter A , Blanco C , Sicard A M
Published: January 2020

Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected.
Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination
Authors: Beck E , Strohmaier K
Published: January 2020

The VP1-coding regions of foot-and-mouth disease virus strains from 18 recent European outbreaks and of 9 strains isolated more than 20 years ago and used in part as vaccines were determined by direct cDNA sequencing. Comparison of the sequences revealed that most of the isolated outbreak viruses are closely related to the vaccine strains used. Isolates from the Italian epizootic of 1984 to 1985 correspond, for example, to the vaccine strain A5 Parma 62; the outbreak in 1984 in Bernbeuren, Federal Republic of Germany, was induced by A5 Allier 60; outbreaks in 1982 in Funen, Denmark, and in Murchin, German Democratic Republic, were caused by O1 Lausanne 65. Viruses isolated during the 1983 Iberian epizootic show a close relationship to the vaccine strain A5 Allier 60 but were probably derived from another not yet identified vaccine strain from Spain. Only two minor outbreaks in the Federal Republic of Germany, A Aachen in 1976 and O Wuppertal in 1982, did not correspond to the classical European strains but were obviously introduced from outside. We suggest that nucleotide sequence analysis should be used as a standard method of diagnosis, because when compared with other techniques it more clearly reveals the origin and course of epizootics and offers the possibility of preventing further outbreaks.
Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product
Authors: Phelps W C , Howley P M
Published: January 2020

We identified a conditional transcriptional enhancer in the long control region (LCR) of human papillomavirus type 16 (HPV-16). This conditional enhancer requires activation in trans by a product of the viral early-region open reading frames (ORFs). Primer extension analysis of chloramphenicol acetyltransferase RNA isolated from transiently transfected CV-1 cells demonstrated that trans-activation of the HPV-16 LCR enhancer operated at the transcriptional level. Mutational analysis of the early ORFs demonstrated that the conditional enhancer of the LCR was trans-activated by the product of the E2 ORF. The E2 gene product of bovine papillomavirus type 1, which can trans-activate the conditional enhancer in the bovine papillomavirus type 1 LCR, was also capable of trans-activating the E2-responsive enhancer of HPV-16. The activity of the HPV-16 LCR enhancer was also assayed in two human cervical carcinoma cell lines, HeLa and SiHa, which harbor transcriptionally active, integrated HPV-18 and HPV-16 DNA sequences, respectively. No endogenous E2 or E2-like activity was detected in either cell line.
Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region
Authors: Bender M A , Palmer T D , Gelinas R E , Miller A D
Published: January 2020

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.
Regulation of transcription of the Xp10 genome in bacteriophage-infected Xanthomonas campestris pv. oryzae
Authors: Liao Y D , Tu J , Kuo T T
Published: January 2020

Results of in vivo studies showed that the transcription of the Xp10 genome in Xp10-infected cells shifted from rifampin sensitivity to rifampin resistance. Results of in vitro studies showed that a rapid reduction of rifampin-sensitive RNA polymerase activity coincided with a rapid increase of rifampin-resistant RNA polymerase activity in cell extracts with time after infection. Host and Xp10-encoded RNA polymerases were purified, and the transcripts from these two enzymes were hybridized to the restriction fragments of Xp10 DNA. The RNA probe generated by host RNA polymerase hybridized strongly to the leftmost 25% of Xp10 DNA and weakly to the rightmost 75% of Xp10 DNA. The RNA probe generated by Xp10 RNA polymerase hybridized strongly to the rightmost 75% of Xp10 DNA and weakly to the leftmost 25% of Xp10 DNA. Studies with 32P-labeled RNA isolated at various intervals after infection did not reveal any evidence for early versus late differences in transcription.
Polyomavirus major capsid protein VP1 is modified by tyrosine sulfuration
Authors: Ludlow J W , Consigli R A
Published: January 2020

Polyomavirus was propagated in primary mouse kidney cell monolayers and 35S-sulfate labeled by maintaining the infected cells in serum-free Eagle medium supplemented with 35S-labeled sodium sulfate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CsCI gradient-purified 35S-sulfate-labeled virions followed by fluorography indicated that the polyomavirus-coded major capsid protein VP1 incorporated this radiolabel. Two-dimensional gel electrophoresis followed by fluorography revealed 35S-sulfate incorporation into only two of the six VP1 isoelectric species (E and F). Amino acid analysis of 35S-sulfate labeled VP1 by enzymatic hydrolysis followed by two-dimensional thin-layer electrophoresis revealed the presence of 35S-sulfate-labeled tyrosine-O-sulfate.
Intratypic recombination of polioviruses: evidence for multiple crossing-over sites on the viral genome
Authors: Agut H , Kean K M , Bellocq C , Fichot O , Girard M
Published: January 2020

Intratypic recombinant polioviruses were isolated from cells that were coinfected with two temperature-sensitive (ts) mutants of poliovirus type 1, ts035Gr and ts247. After phenotypic characterization of these recombinants, their proteins were studied by polyacrylamide gel electrophoresis, and their genomes were analyzed by RNase T1 fingerprinting and partial nucleotide sequencing. Segregation of specific phenotypic and biochemical characteristics inherited from the parental viruses demonstrated that crossing-over could occur in at least four distinct regions of the genome. Possible mechanisms for recombination are discussed.
High-frequency homologous recombination in vaccinia virus DNA
Authors: Ball L A
Published: January 2020

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.