bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the binding proteins and activity of the long terminal repeat of Moloney murine leukemia virus during differentiation of mouse embryonal carcinoma cells
Authors: Tsukiyama T , Niwa O , Yokoro K
Published: January 2020

Mouse embryonal carcinoma (EC) cell lines were established which carry the stably integrated chloramphenicol acetyltransferase (CAT) gene under the control of the transcriptional elements of the long terminal repeat (LTR) of Moloney murine leukemia virus. The activity of three elements of the stably integrated LTR was analyzed in undifferentiated EC cells (stable CAT assay). Results of the study are summarized as follows. (i) In the stable assay, the promoter region of the LTR was inactive in undifferentiated ECA2 and F9 cells, and the level of the activity was 10(-4) of that in NIH 3T3 cells. (ii) In contrast to the results of the transient assay, the enhancer was active in undifferentiated ECA2 cells and in F9 cells. It activated CAT activity more than 60-fold and about 8-fold in ECA2 cells and F9 cells, respectively. (iii) Suppression by ELP, the embryonal LTR-binding protein, was more pronounced in the stable assay than in the transient assay. These data suggest that, when compared with NIH 3T3 cells, a major factor for the inactivity of the LTR in EC cells is the inefficiency of the promoter in this assay. Transcriptional activity of the LTR was analyzed during the differentiation of EC cells. In the case of ECA2 cells, the magnitude of activation by the enhancer did not change during differentiation. The activity of the promoter increased about 10-fold, and the suppression by ELP became negligible 4 days after the induction of differentiation. Upon differentiation of F9 cells, the activity of the enhancer increased more than 300-fold, but the promoter remained inactive. The pattern of LTR-binding proteins also varied during the differentiation of EC cells. Our present data suggest that the activity of LTR elements as assayed by the stable assay differs from the activity as assayed by the transient assay. It also indicates that the activity of these elements exhibits cell-type-specific changes during the differentiation of EC cells.
Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos
Authors: Mélin F , Kemler R , Kress C , Pinon H , Blangy D
Published: January 2020

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.
In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release
Authors: Liljeström P , Lusa S , Huylebroeck D , Garoff H
Published: January 2020

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.
The cellular proto-oncogene product Myb acts as transcriptional activator of the long terminal repeat of human T-lymphotropic virus type I
Authors: Dasgupta P , Reddy C D , Saikumar P , Reddy E P
Published: January 2020

The proto-oncogene c-myb encodes a nuclear transcription factor that binds to DNA in a sequence-specific manner and activates transcription of several viral and cellular genes. Expression of the c-myb gene is induced in mitogen- and/or antigen-stimulated T lymphocytes, which are also the preferential target cells of human T-lymphotropic virus type I (HTLV-I) in vivo and in vitro. We report here that Myb binds to the HTLV-I long terminal repeat (LTR) in four different regions in a sequence-specific manner. Electrophoretic mobility shift assay using labeled LTR fragments as well as labeled double-stranded oligonucleotides show that there are two high-affinity and two low-affinity Myb-binding sites present in the HTLV-I LTR. DNase I footprinting analysis and oligonucleotide competition experiments indicate that this binding is sequence specific. Cotransfection experiments in HeLa cells, using a Myb expression vector and chloramphenicol acetyltransferase reporter gene linked to the HTLV-I LTR, show that Myb activates HTLV-I LTR-mediated transcription by a factor of four-to sixfold. Thus, in HTLV-I-infected T cells, Myb protein binding to the HTLV-I LTR may constitute one of the signal that regulate HTLV-I transcription in vivo.
Tunicamycin treatment of CHO cells abrogates multiple blocks to retrovirus infection, one of which is due to a secreted inhibitor
Authors: Miller D G , Miller A D
Published: January 2020

Chinese hamster ovary (CHO) cells are resistant to infection by all of the major classes of murine retroviruses and are partially resistant to infection by gibbon ape leukemia virus. Treatment of CHO cells with the glycosylation inhibitor tunicamycin rendered these cells susceptible to infection by retroviral vectors with ecotropic, xenotropic, and amphotropic host ranges and increased the titer of gibbon ape leukemia virus pseudotyped vectors 10-fold. Vectors having a polytropic host range did not infect CHO cells in the presence or absence of tunicamycin, showing that the effect of tunicamycin was specific and related to the pseudotype of the vector. We present evidence for three mechanisms of resistance to infection: lack of viral receptors on CHO cells, the presence of nonfunctional receptors which can be made functional by treatment with tunicamycin, and the secretion of a protein factor that blocks retroviral infection of CHO cells. Several criteria indicate that the secreted inhibitor is not an interferon, and secretion of this factor was not detected in several other cell lines that were examined.
Neutralizing antibodies modulate replication of simian immunodeficiency virus SIVmac in primary macaque macrophages
Authors: McEntee M F , Zink M C , Anderson M G , Farzadegan H , Adams R J , Kent K A , Stott E J , Clements J E , Narayan O
Published: January 2020

Cultured macaque macrophages are permissive for the replication of SIVmac251, and inoculation with virus is followed by the production of viral p27. Neutralizing macaque polyclonal and murine monoclonal antibodies preincubated with the virus prevented infection but did not prevent cytopathic virus replication when added more than 3 days after inoculation with virus. However, application of the neutralizing antibodies to macrophages 24 h after inoculation with virus resulted in sustained, low-level production of viral antigen. Cell lysates and individual macrophages from treated cultures contained less viral protein by Western blot (immunoblot) and immunocytochemistry than untreated controls. In situ hybridization and polymerase chain reaction procedures for detecting and estimating relative amounts of viral RNA and DNA showed that both viral nucleic acids failed to increase beyond the levels obtained before the addition of neutralizing antibodies. The data suggest that macrophages may need to be infected with a minimum threshold of virus particles in order to reach their full potential for virus replication and that their exposure to neutralizing antibodies prior to reaching this threshold resulted in limited virus replication.
Negative regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells via a retinoic acid response element
Authors: Kralli A , Ge R , Graeven U , Ricciardi R P , Weinmann R
Published: January 2020

In cells transformed by the highly oncogenic adenovirus type 12 (Ad12), the viral E1A proteins mediate transcriptional repression of the major histocompatibility class I genes. In contrast, class I transcription is not reduced in cells transformed by the nononcogenic Ad5. The decreased rate of class I transcription is, at least in part, the result of a reduced major histocompatibility complex class I enhancer activity in Ad12-transformed cells and correlates with an increase in the levels of a DNA-binding activity to the R2 element of the enhancer (R. Ge, A. Kralli, R. Weinmann, and R. P. Ricciardi, J. Virol. 66:6969-6978, 1992). Employing transient transfection assays, we now provide direct evidence that the R2 element can confer repression in Ad12- but not Ad5-transformed cells. Repression by R2 was observed only in the presence of the positive enhancer element R1 and was dependent on (i) the number of the R2 elements and (ii) the relative arrangement of R2 and R1 elements. The putative R2-binding repressor protein, R2BF, was similar in molecular weight and binding specificity to members of the thyroid hormone/retinoic acid (RA) receptor family. RA treatment abrogated the R2-mediated repression in Ad12-transformed cells and had no effect on the activity of R2/R1-containing promoters in Ad5-transformed cells. These results are consistent with the presence of an R2-binding repressor in Ad12-transformed cells. In the absence of RA, the repressor compromises enhancer activity by interfering with the activity of the positive cis element R1. RA treatment of Ad12-transformed cells may render the repressor inactive.
The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome
Authors: Smiley J R , Lavery C , Howes M
Published: January 2020

We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.
In vitro resolution of adeno-associated virus DNA hairpin termini by wild-type Rep protein is inhibited by a dominant-negative mutant of rep
Authors: Owens R A , Carter B J
Published: January 2020

An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo. We expressed both wild-type (Rep78) and mutant (Rep78NTP) proteins in two helper-free expression systems consisting of either recombinant baculoviruses in insect cells or the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cell transient transfections. We analyzed nuclear extracts from both expression systems for the ability to complement uninfected HeLa cell cytoplasmic extracts in an in vitro terminal resolution assay in which a covalently closed AAV terminal hairpin structure is converted to an extended linear duplex. Although both Rep78 and Rep78NTP bound to AAV terminal hairpin DNA in vitro, Rep78 but not Rep78NTP complemented the terminal resolution assay. Furthermore, Rep78NTP was trans dominant for AAV terminal resolution in vitro. We propose that the dominant-negative replication phenotype of AAV genomes carrying the K340H mutation is mediated by mutant Rep proteins binding to the terminal repeat hairpin.
Effect of zinc ions on the biochemical behavior of simian virus 40 small-t antigen expressed in bacteria
Authors: Goswami R , Turk B , Enderle K , Howe A , Rundell K
Published: January 2020

The simian virus 40 small-t antigen contains 10 cysteine residues, 6 of which are organized in two CysXCysXXCys clusters. Mutation of individual Cys residues in the two clusters or mutation of specific residues found between these clusters causes pronounced instability of the protein in animal cells. Protein instability correlates with failure of the bacterially expressed mutant proteins to bind zinc ions, an interaction which allows purification of large amounts of small-t antigen in monomeric form.
Ar+ plasma-induced damage to DNA in bacteriophage lambda: implications for the arrangement of DNA in the phage head
Authors: Mendelson E C , Newcomb W W , Brown J C
Published: January 2020

Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass. The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell. Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA. Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined. Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population. After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction. Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end. In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population. In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined. The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome. Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.
Differentiation-dependent human immunodeficiency virus long terminal repeat regulatory elements active in human teratocarcinoma cells
Authors: Zeichner S L , Hirka G , Andrews P W , Alwine J C
Published: January 2020

We have examined the transcriptional utilization of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) under differentiating conditions by using the embryonal carcinoma cell line NTERA-2. NTERA-2 cells undergo two distinct pathways of terminal differentiation, to a neuronal phenotype in response to retinoic acid and to a nonneuronal phenotype in response to hexamethylene bisacetamide. To identify LTR regulatory elements active in each cell type we used a set of HIV LTR linker substitution mutants, which contain mutations that progressively replace adjacent 18-bp segments across the U3 region and into the R region (between nucleotides -453 and +15 relative to the transcription start site). Although each differentiating cell type showed utilization of expected key elements (e.g., NF-kappa B, SP1, TATA) in the 3' portion of the LTR (+1 to -112), the data indicated differentiation-dependent differences in the utilization of these elements. In addition, regions showing dramatic differentiation-dependent effects were detected in the 5' portion of the LTR (-112 to -453), in positions where transcription control elements have not been described previously. The marked differences in the sets of LTR regulatory elements required by each cell type indicate that the LTR can function under a variety of differentiation conditions. Together with previous findings, the data suggest that the complexity of the HIV LTR for transcriptional control is much greater than was previously thought and that the LTR maintains elements which facilitate transcription in many cell types.
Evolutionary insights on the origin of human T-cell lymphoma/leukemia virus type I (HTLV-I) derived from sequence analysis of a new HTLV-I variant from Papua New Guinea
Authors: Sherman M P , Saksena N K , Dube D K , Yanagihara R , Poiesz B J
Published: January 2020

Recent studies have established the presence of human T-cell lymphoma/leukemia virus type I (HTLV-I) in Melanesia. An HTLV-I strain, PNG-1, has now been isolated from a healthy member of the Hagahai, a remote, recently contacted group in Papua New Guinea. To further characterize PNG-1, we employed polymerase chain reaction amplification with subsequent cloning and sequencing of amplified products. Sequence analyses of amplified regions of pol, env, and pX genes of this variant indicate marked heterogeneity (approximately 7%) from prototype HTLV-I. Based on available sequence data, PNG-1 is distinct from all other known HTLV-I strains and diverges from the common ancestor of HTLV-I prior to prototype isolates. The data also suggest, therefore, that HTLV-I originated in the Indo-Malay region rather than Africa.
Hemagglutinin-esterase-specific monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus
Authors: Yokomori K , Baker S C , Stohlman S A , Lai M M
Published: January 2020

Some of mouse hepatitis virus strains contain an optional envelope glycoprotein, hemagglutinin-esterase (HE) protein. To understand the functional significance of this protein, monoclonal antibodies (MAbs) specific for this protein were generated and used for passive immunization of mice. None of these MAbs showed any virus-neutralizing activity in vitro; however, mice passively immunized with the purified MAbs were protected from lethal infection by the JHM strain of mouse hepatitis virus. Passive immunization altered the pathogenicity such that the virus caused subacute and chronic demyelination instead of acute lethal encephalitis. Virus titers in the brains of the immunized mice were significantly lower than those for the nonimmunized control mice, suggesting that the virus replication or spread was inhibited. In addition, histopathological analysis indicated that the spread of virus in the brain and spinal cord was significantly inhibited in the immunized mice. Furthermore, the mononuclear cell infiltration in the immunized mice appeared earlier than in the nonimmunized mice, suggesting that the exogenous antibody might have activated host immune responses, and thus facilitated clearance of the virus or virus-infected cells. The same protective effects were observed for both JHM(2) and JHM(3) viruses, which expressed different amounts of the HE protein. In contrast, mice infected with At11f, a variant of JHM which does not express the HE protein, were not protected by these MAbs, suggesting that protection was mediated by the specific interaction between the MAb and the HE protein. Thus, the mechanism of protection by the exogenous HE-specific MAbs may represent the early activation of innate immune mechanisms in response to the interaction between the MAbs and the HE protein.
Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells
Authors: Kannagi M , Shida H , Igarashi H , Kuruma K , Murai H , Aono Y , Maruyama I , Osame M , Hattori T , Inoko H
Published: January 2020

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sjögren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.
Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter
Authors: Rubinstein S J , Hammerle T , Wimmer E , Dasgupta A
Published: January 2020

In HeLa cells, RNA polymerase I (Pol I)-mediated transcription is severely inhibited soon after infection with poliovirus. We have developed a gel retardation assay to analyze DNA-protein complexes formed at the Pol I promoter. We show here that two complexes (A and C) formed by nuclear extracts from uninfected cells disappear after infection of cells with poliovirus. In contrast, a new, rapidly migrating complex (D) is formed in virus-infected cell extract. This change in the mobility of gel-retarded complexes correlates well with the kinetics of inhibition of rRNA transcription in virus-infected cells. Incubation of nuclear extracts from mock-infected cells with bacterially expressed, purified poliovirus protease 3C results in the disappearance of complexes A and C with concomitant generation of complex D. A partially purified transcription factor fraction derived from uninfected cells that contains complex A is able to restore Pol I transcription when added to virus-infected cell extracts, suggesting that this complex plays an important role in Pol I transcription. These results suggest that poliovirus proteinase 3C may have an important role in the shutoff of Pol I transcription in cells infected with poliovirus.
Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements
Authors: May E , Omilli F , Borde J , Scieller P
Published: January 2020

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.
Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype)
Authors: Grigera P R , Keil W , Wagner R R
Published: January 2020

Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which ostensibly determines the conformational structure of the VSV-New Jersey G protein required for presentation of the major discontinuous epitopes recognized by neutralizing MAbs.
Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1
Authors: Haynes J I , Consigli R A
Published: January 2020

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.
Activation of simian virus 40 transcription in vitro by T antigen
Authors: Coulombe J , Berger L , Smith D B , Hehl R K , Wildeman A G
Published: January 2020

Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues. Late transcription is activated by TAg, even in the absence of viral DNA replication. We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system. In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters. When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected. Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other. Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription. An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein. Structural features of this region that might be important are discussed.
Evolution of the V3 envelope domain in proviral sequences and isolates of human immunodeficiency virus type 1 during transition of the viral biological phenotype
Authors: Kuiken C L , de Jong J J , Baan E , Keulen W , Tersmette M , Goudsmit J
Published: January 2020

The third variable domain (V3) of the envelope gene of human immunodeficiency virus type 1 contains a major neutralization epitope and determinants of syncytium-inducing (SI) capacity and replication rate (reviewed by J. P. Moore and P. L. Nara, AIDS Suppl. 2:S21-S33, 1991). Sequences were generated from DNA of samples taken 3 months apart over a period of 24 and 30 months from peripheral blood mononuclear cells (PBMC) of two individuals, both before and after cocultivation with uninfected donor PBMC. The isolated virus shifted from the non-syncytium-inducing (NSI) phenotype to the SI phenotype during the study period. This shift was associated with distinct changes in the V3 domain in both patients. The association of the phenotype shift with the V3 sequence changes was confirmed by construction of viruses with chimeric V3 loops. The shift from NSI- to SI-associated V3 variants was also seen in the uncultured PBMC of both patients, but not until 3 and 9 months after the detection of SI virus in culture. In the samples of uncultured PBMC DNA, several subgroups of sequences were found, indicating that the process of evolution may not be gradual and that several distinct populations can coexist. The paucity of intermediate sequences indicated that strong selection pressure was exerted on this part of the envelope. The early emergence of disease-associated SI variants in cultured material indicates that virus culture may have relevance for the in vivo situation.
Postinfection treatment with antiviral serum results in survival of neural cells productively infected with virulent poliovirus
Authors: Tolskaya E A , Ivannikova T A , Kolesnikova M S , Drozdov S G , Agol V I
Published: January 2020

The death of human neuroblastoma cells undergoing productive infection with virulent poliovirus was prevented by addition of antiserum against the virus a few hours after the onset of infection; this treatment, however, did not prevent reproduction of the virus. Despite the presence of the viral antigen, the cells retained the ability to divide. Upon further cultivation in the absence of antiserum, the cells developed specific postinfection immunity or resistance to superinfection with poliovirus.
Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli
Authors: Harmon S A , Updike W , Jia X Y , Summers D F , Ehrenfeld E
Published: January 2020

To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987. Very little further cleavage of the released P3 protein was detected. When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al. (J. Virol. 61:50-59, 1987). The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C. Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.
Expression of the influenza A virus M2 protein is restricted to apical surfaces of polarized epithelial cells
Authors: Hughey P G , Compans R W , Zebedee S L , Lamb R A
Published: January 2020

The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of other influenza virus proteins and that M2 contains the structural features required for apical transport in polarized epithelial cells. The ultrastructural localization of the M2 protein in influenza virus-infected MDCK cells was investigated by immunoelectron microscopy using M2 antibody and a gold conjugate. In cells in which extensive virus budding was occurring, the apical cell membrane was labeled with gold particles evenly distributed between microvilli and the surrounding membrane. In addition, a significant fraction of the M2 label was apparently associated with virions. A monoclonal antibody specific for HA demonstrated a similar labeling pattern. These results indicate that M2 is localized in close proximity to budding and assembled virions.
Differentiated liver cell specificity of the second enhancer of hepatitis B virus
Authors: Yuh C H , Ting L P
Published: January 2020

Hepatitis B virus is a hepatotropic virus. Its replication and gene expression are mainly restricted to hepatocytes in the infective process. The viral gene expression thus provides a unique system with which to study the control of tissue-specific gene expression. We have previously reported the identification and characterization of the second enhancer (enhancer II) of hepatitis B virus. In this report, we further demonstrate that the minimal functional constituents of the second enhancer, box alpha and box beta, display liver cell and differentiation state specificity. Moreover, box alpha exhibits the same liver cell and differentiation state specificity when functioning as an upstream regulator for the basal core promoter. Gel shift experiments reveal a unique box alpha-binding protein, protein a, which is present only in differentiated liver cells, where enhancer II is functional. The converse is true for another box alpha-binding protein, protein f, which is present only in poorly differentiated liver cells and nonliver cells. The simplest hypothesis that explains these results is that protein a activates and/or protein f suppresses the enhancer and upstream regulator functions. Although C/EBP is a candidate for a transcription factor that interacts with box alpha or box beta, none of the binding factors identified in the gel shift assays, including protein a and protein f, is likely to be C/EBP because they differ from C/EBP in heat lability and sequence preference.
Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses
Authors: Clarke D K , Duarte E A , Moya A , Elena S F , Domingo E , Holland J
Published: January 2020

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.
Inhibition of herpes simplex virus type 1 DNA polymerase activity by peptides from the UL42 accessory protein is largely nonspecific
Authors: Owsianka A M , Hart G , Murphy M , Gottlieb J , Boehme R , Challberg M , Marsden H S
Published: January 2020

To identify regions in the UL42 protein of herpes simplex virus type 1 which affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acids of the UL42 protein were synthesized and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: formation of full-length double-stranded M13 molecules and rate of incorporation of deoxyribonucleoside triphosphates. Peptides from five noncontiguous regions of the UL42 protein were found to inhibit herpes simplex virus type 1 polymerase activity in both the presence and absence of UL42 protein. The most active peptides from each region correspond to amino acids 23 to 38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47, and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase.
Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA
Authors: Brandsma J L , Xiao W
Published: January 2020

The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.
Cytopathogenic effect in cardiac myocytes but not in cardiac fibroblasts is correlated with reovirus-induced acute myocarditis
Authors: Baty C J , Sherry B
Published: January 2020

A panel of reovirus strains was used to compare myocarditic potential with induction of cytopathic effect in primary cardiac myocyte and cardiac fibroblast cultures. The results suggest that viral cytopathogenicity in cardiac myocytes, but not in cardiac fibroblasts, is a determinant of reoviral myocarditis.
Individual repeats of Drosophila Myb can function in transformation by v-Myb
Authors: Bin X , Lipsick J S
Published: January 2020

The v-Myb protein binds to specific DNA sequences and can regulate gene expression. The DNA-binding domain of v-Myb contains the second and third of the three highly conserved tandem repeats found in c-Myb. In general, the ability of mutant forms of v-Myb to transform correlates with their ability to trans activate transcription. Two mutations within the DNA-binding domain of v-Myb which preserve DNA binding in vitro but fail to trans activate or transform have been described. These results suggested that this highly conserved domain might function in specific protein-protein interactions, as well as in DNA binding. We therefore tested the ability of a related protein domain from Drosophila melanogaster to substitute functionally for the homologous region of v-Myb. We found that either the second or third repeat of Drosophila Myb, but not both, could function in trans-activation and transformation by v-Myb. The hybrid containing both the second and third repeats of Drosophila Myb bound to DNA but failed to trans activate transcription either in the context of v-Myb or as a v-Myb-VP16 fusion protein. These results demonstrate that although the protein-DNA contacts made by the Myb repeats have been conserved during the evolution of animals, the protein-protein interactions have diverged.
Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion
Authors: Horton H M , Burand J P
Published: January 2020

This research provides the first evidence for specific receptor binding of polyhedron-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a nonendocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting analysis was used to investigate the specificity of binding between the PDV phenotype of Lymantria dispar nuclear polyhedrosis virus (LdNPV) and host membranes. Fluorescein isothiocyanate-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line IPLB-LdEIta and to L. dispar BBMV. The IPLB-LdEIta cell line was found to possess approximately 10(6) PDV-specific receptor sites per cell. Excess levels of unlabeled PDV were highly efficient in competing with fluorescein isothiocyanate-labeled PDV for limited receptor sites, further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV was used to identify PDV fusion to host cells and BBMV. Direct membrane fusion of PDV occurred at 27 degrees C to both target membranes as well as at 4 degrees C at approximately 55% of the levels achieved at 27 degrees C. Viral fusion to BBMV occurred throughout the pH range of 4 to 11, with dramatically increased fusion levels (threefold) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture assays.