bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction
Authors: Dimitrov D S , Broder C C , Berger E A , Blumenthal R
Published: January 2020

Calcium ions are required for fusion of a wide variety of artificial and biological membranes. To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1. Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy. Syncytia did not form in solutions without calcium ions. Addition of calcium ions partially restored the formation of syncytia. EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] blocked syncytium formation in culture media containing calcium ions. Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions. Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM. Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+. Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+. We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.
A single amino acid in the PB2 gene of influenza A virus is a determinant of host range
Authors: Subbarao E K , London W , Murphy B R
Published: January 2020

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.
T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: relevance to vaccine development
Authors: Gatei M H , Good M F , Daniel R C , Lavin M F
Published: January 2020

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and sheep and may provide a model for studying human leukemia. Cell-mediated immune mechanisms may play a major role in protection against BLV infection. We describe here for the first time the identification of proliferative (CD4) and cytotoxic T-lymphocyte (CD8) epitopes of the gp51 envelope (env) protein of BLV. This protein and a recombinant form expressed by a vaccinia virus construct have been shown to be potential vaccine candidates. A complete series of overlapping peptides, 20 amino acids in length, was prepared to identify epitopes from gp51. These peptides were tested for the ability to elicit peripheral blood lymphocyte proliferation and cytotoxic T-lymphocyte responses in infected and uninfected cattle and sheep. Peptides 51-70 and 61-80 produced a proliferative response in lymphocytes from only uninfected animals (both sheep and cattle), and this was shown by T-cell subset deletion to be a CD4-mediated response. Seven BLV-infected sheep did not show a response to either peptide. Cytotoxic T-lymphocyte activity, however, was associated only with peptides 121-140 and 131-150. In this case, the response was demonstrated to be CD8 dependent and was found only in BLV-infected animals (sheep). Knowledge of the location of these T-cell recognition domains will complement data available on B-cell epitopes in gp51 and may be useful in the design of a subunit vaccine.
Isolation of a chicken gene that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses
Authors: Young J A , Bates P , Varmus H E
Published: January 2020

We used a genetic strategy to isolate the chicken gene believed to encode the receptor for subgroup A avian leukosis and sarcoma viruses (ALSV-A). Chicken genomic DNA was transfected into monkey COS-7 cells, and two independent primary transfectants susceptible to ALSV-A infection were identified by using ALSV-A vectors containing a hygromycin B resistance gene. A second round of transfection and selection in mouse BALB/3T3 fibroblasts again led to isolation of a transfectant susceptible to infection by ALSV-A. Plasmid DNA sequences linked to chicken DNA during the primary transfection segregated with chicken DNA in the secondary transfectant and served as a molecular tag to clone the gene conferring susceptibility. Expression of the cloned gene in mouse BALB/3T3 cells conferred susceptibility to infection by ALSV-A but not by ALSV-B. Therefore the cloned gene most probably represents the tv-a locus, the genetically defined receptor gene for ALSV-A.
Mutation of a cysteine residue in polyomavirus middle T antigen abolishes interactions with protein phosphatase 2A, pp60c-src, and phosphatidylinositol-3 kinase, activation of c-fos expression, and cellular transformation
Authors: Glenn G M , Eckhart W
Published: January 2020

Polyomavirus middle T antigen (MT) interacts with several cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src (and the related kinases c-fyn and c-yes), and phosphatidylinositol-3 kinase. We made a single point mutation in MT, changing a conserved cysteine residue at position 120 to tryptophan, and characterized the biochemical and biological properties of the mutant (C120W) protein. The mutant MT protein does not associate with PP2A, pp60c-src, or phosphatidylinositol-3 kinase as judged by coimmunoprecipitation and associated phosphatase or kinase activity. The C120W mutant is defective in activation of c-fos expression and in morphological transformation of NIH 3T3 cells.
Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes
Authors: Hijikata M , Shimizu Y K , Kato H , Iwamoto A , Shih J W , Alter H J , Purcell R H , Yoshikura H
Published: January 2020

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.
Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation
Authors: Tomkinson B , Robertson E , Kieff E
Published: January 2020

Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or -3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformation-defective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation.
Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes
Authors: Closs E I , Rinkes I H , Bader A , Yarmush M L , Cunningham J M
Published: January 2020

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.
Structure-function analysis of the maf oncogene product, a member of the b-Zip protein family
Authors: Kataoka K , Nishizawa M , Kawai S
Published: January 2020

The v-maf oncogene, identified as the transforming gene of the avian retrovirus AS42, encodes a protein containing a b-Zip motif. From this structural feature, the v-Maf protein was expected to form a dimer and function as a nuclear DNA-binding protein. In this study, we demonstrate that this protein indeed localizes predominantly in the nucleus and forms a homodimer through its leucine zipper structure. To delineate the structural requirement for the transforming activity, we constructed and characterized a panel of v-maf mutants harboring various deletions or point mutations. A region of about 100 amino acid residues located near its carboxyl terminus, which contains the b-Zip motif, was found to be essential for the basal transforming activity of v-Maf on chicken embryo fibroblasts. On the other hand, the amino-terminal two-thirds of the v-Maf protein seems to play a role in potentiating the transforming activity of v-Maf. It was also found that the c-maf proto-oncogene, without any structural modification in its protein-coding region, could transform cells as efficiently as could the v-maf oncogene when transduced by a retroviral vector. Thus, it is probably deregulated expression that makes the v-maf gene oncogenic. In addition, we discovered one point mutation, altering the structure of the b-Zip domain, which further enhances the transforming activity of the v-maf oncogene. Such mutant will be useful in exploring the mechanism of action of the Maf protein.
The blocks to human immunodeficiency virus type 1 Tat and Rev functions in mouse cell lines are independent
Authors: Winslow B J , Trono D
Published: January 2020

Rodent cells present two blocks precluding the expression of the human immunodeficiency virus type 1 (HIV-1) genome. First, the viral protein Tat is only poorly active in these cells. Second, when the HIV-1 provirus is integrated in the genome of mouse cells, it electively fails to express the viral structural proteins, indicating a block to Rev action. Both defects can be complemented by fusion of the infected mouse cells with uninfected human cells. Because the production of high levels of Rev is dependent on Tat-mediated transactivation and because both Tat and Rev bind the viral transcript, it has been hypothesized that the two blocks found in rodent cells might be linked. In the present work, we demonstrate that overexpression of Rev in mouse cell lines does not relieve their block in HIV-1 structural-gene expression. In addition, we show that this defect is also present in human-mouse cell hybrids which contain human chromosome 12 and support Tat function. On that basis, we conclude that the blocks to HIV-1 Tat and Rev action in mouse cell lines are independent and result from the absence of distinct cellular elements that are critical for HIV-1 gene expression.
The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments
Authors: Koralnik I J , Fullen J , Franchini G
Published: January 2020

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.
Sequence specificity of the core-binding factor
Authors: Melnikova I N , Crute B E , Wang S , Speck N A
Published: January 2020

The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.
Apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus
Authors: Ohno K , Nakano T , Matsumoto Y , Watari T , Goitsuka R , Nakayama H , Tsujimoto H , Hasegawa A
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) induced morphologic changes such as chromatin condensation and cell shrinkage in a feline fibroblastic cell line (CRFK) chronically infected with feline immunodeficiency virus (FIV) but not in uninfected CRFK cells. DNA extracted from TNF-alpha-treated CRFK cells infected with FIV showed a ladder of nucleosomal DNA, indicating that this cytocidal effect by TNF-alpha was due to programmed cell death, or apoptosis. These findings may have implications for understanding the pathogenesis of FIV infection and for the design of specific therapeutic strategies for AIDS in humans as well as cats.
Isolation and genetic characterization of ethanol-resistant reovirus mutants
Authors: Wessner D R , Fields B N
Published: January 2020

To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.
Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase
Authors: Grandgenett D P , Inman R B , Vora A C , Fitzgerald M L
Published: January 2020

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.
Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein
Authors: Cao J , Bergeron L , Helseth E , Thali M , Repke H , Sodroski J
Published: January 2020

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp41 human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp41 ectodomain.
Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein
Authors: Ojala P M , Juuti J T , Bamford D H
Published: January 2020

Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity. We have produced and characterized a panel of 20 P4-specific monoclonal antibodies. Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes. The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.
Identification of an amino acid that defines the fusogenicity of mumps virus
Authors: Tanabayashi K , Takeuchi K , Okazaki K , Hishiyama M , Yamada A
Published: January 2020

Recombinant cDNA clones representing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of two mumps virus strains different in fusogenicity were constructed. Upon transfection of COS7 cells, extensive cell fusion was observed only when cells expressed the F protein of the fusing strain together with the HN protein derived from either strain. Mutational analyses further showed that the amino acid at position 195 of the F protein plays a critical role in determining the extent of cell fusion induced by mumps virus, since replacement of Ser-195 by Tyr significantly reduced the fusion inducibility of otherwise fusion-competent F protein.
Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture
Authors: Sánchez-Palomino S , Rojas J M , Martínez M A , Fenyö E M , Nájera R , Domingo E , López-Galíndez C
Published: January 2020

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.
Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection
Authors: Söderberg C , Larsson S , Bergstedt-Lindqvist S , Möller E
Published: January 2020

The identity of cells responsible for transmission of human cytomegalovirus (HCMV) in blood products or bone marrow transplants is unknown. We have tested the capacity of HCMV to in vitro infect human peripheral blood mononuclear cells (PBMC) from healthy donors and found that certain PBMC are permissive to HCMV infection. In vitro-infected viable cells were double stained for surface expression of different HMCV proteins and for cell-type-specific antigens to allow the identification of sensitive cells. All analysis were performed on viable cells, using HCMV-specific monoclonal antibodies and automated flow cytofluorimetry. PBMC were infected either with the laboratory-adapted HCMV strain AD169 or with a virus isolate obtained from a viremic patient. Up to 25% of all PBMC could express the major immediate-early antigen as well as the pp65 antigen, known at the lower matrix protein. Infected cells were mainly CD14+ monocytes, but also a small population of large CD8+ cells were susceptible to HCMV infection. CD19+ B lymphocytes were resistant to HCMV infection. Different populations of infected cells were enriched by using Dynabeads coated with cell-type-specific antibodies, and the presence of infectious virus was demonstrated by incubating the selected and sonicated cell material on human fibroblasts. Only material from infected monocytes and from CD3+ CD8+ cells gave rise to HCMV-specific plaques. The presence of HCMV mRNA as a sign of active viral transcription of the major immediate-early and late pp150 genes in infected cells was demonstrated by using nested reversed polymerase chain reaction. A common denominator was found for all cells that could be infected with HCMV. The CD13 antigen, a 130- to 150-kDa integral membrane protein identical to the enzyme aminopeptidase N, was expressed on all HCMV-permissive cells.
Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion
Authors: Griffiths J C , Harris S J , Layton G T , Berrie E L , French T J , Burns N R , Adams S E , Kingsman A J
Published: January 2020

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.
Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef
Authors: Schwartz O , Rivière Y , Heard J M , Danos O
Published: January 2020

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.
Processing of the envelope glycoproteins of pestiviruses
Authors: Rümenapf T , Unger G , Strauss J H , Thiel H J
Published: January 2020

The genomic RNA of pestiviruses is translated into a large polyprotein that is cleaved into a number of proteins. The structural proteins are N terminal in this polyprotein and include three glycoproteins called E0, E1, and E2 on the basis of the order in which they appear in the polyprotein. Using pulse-chase experiments, we show that a pestiviral glycoprotein precursor, E012, is formed that is processed into E0, E1, and E2 in an ordered fashion. Processing is initiated by a nascent cleavage between the capsid and the translocated E012 followed by cleavage at the C terminus of E2. E012 is then rapidly cleaved to form E01 and E2. After E2 is released from the precursor, E01 is processed into E0 and E1. To identify the sites of cleavage, the N termini of the glycoproteins of the pestivirus classical swine fever virus (formerly termed hog cholera virus) were sequenced after expression in the vaccinia virus system. The N termini are Glu-268 for E0 (gp44/48), Leu-495 for E1 (gp33) and Arg-690 for E2 (gp55). The sequences around the cleavage sites capsid/E0 and E1/E2 conform to the rules known for cellular signal proteases, as does the sequence at the presumed C terminus of E2. The sequence upstream of the E0/E1 cleavage site also shows sequence characteristics of signalase processing sites but lacks the typical hydrophobic signal peptide; this cleavage site has characteristics in common with a site in flaviviruses that is also cleaved in a delayed fashion. The absence of any membrane-spanning region results in the shedding of E0 by infected cells, and E0 can be detected in the virus-free supernatant. Comparison of the sequences around the cleavage sites of pestiviruses suggests a general processing scheme for the structural glycoproteins. Comparison of the pesti- and flaviviral structural glycoproteins suggests analogies between E012 and prM-E.
Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation
Authors: Lahser F C , Marsh L E , Hall T C
Published: January 2020

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts. Deletion of single stem-loop structures gave progeny retaining near-wild-type accumulation levels. Constructions representing deletion of two or three stem-loops substantially lowered the accumulation of progeny RNA-3 relative to wild-type levels. RNA-3 mutants bearing deletions of longer sequences or of the entire region (delta PsKs RNA-3) replicated poorly, yielding no detectable RNA-3 or RNA-4 progeny. Levels of RNA-1 and RNA-2, in the presence of a mutant RNA-3, were found to increase relative to the accumulation observed in a complete wild-type transfection. The stability of delta PsKs RNA-3 in protoplasts was somewhat lower than that of wild-type RNA during the first 3 h postinoculation. Little difference in translatability in vitro of wild-type and RNA-3 constructs bearing deletions within the stem-loop region was observed, and Western immunoblot analysis of viral coat protein produced in transfected protoplasts showed that protein accumulation paralleled the amount of RNA-4 message produced from the various sequences evaluated. These results indicate that the RNA-3 pseudoknot region plays a minor role in translational control but contributes substantially to the overall replication of the brome mosaic virus genome.
Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing
Authors: O'Banion M K , Winn V D , Settleman J , Young D A
Published: January 2020

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.
Protective anti-reovirus monoclonal antibodies and their effects on viral pathogenesis
Authors: Tyler K L , Mann M A , Fields B N , Virgin H W
Published: January 2020

We used a recently isolated and characterized panel of monoclonal antibodies (MAbs) specific for cross-reactive determinants on reovirus outer capsid proteins to define mechanisms of antibody-mediated protection in vivo. We studied the capacities of MAbs to protect against lethal infection with reoviruses which differ in site of primary replication, route of spread, and central nervous system tropism. We found the following. (i) MAbs specific for each of the viral outer capsid proteins (sigma 1, sigma 3, and mu 1) and the core spike protein (lambda 2) were protective under certain circumstances. (ii) In vitro properties of MAbs, including isotype, neutralization of viral infectivity, inhibition of virus-induced hemagglutination, and avidity of binding, were poorly predictive of the capacities of MAbs to protect in vivo. (iii) MAbs did not act at a single stage during pathogenesis to mediate protection; instead, protective MAbs were capable of altering a variety of stages in reovirus pathogenesis. (iv) MAbs protective against one reovirus also protected against other reoviruses that utilized different pathogenetic strategies, suggesting that the viral epitope bound by an antibody rather than the pathogenetic strategy employed by the virus is a critical determinant of antibody-mediated protection in vivo. (v) A prominent mechanism of protective MAb action is inhibition of viral spread through nerves from a site of primary replication (e.g., the intestine or muscle tissue) to the central nervous system.
Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus
Authors: La Posta V J , Auperin D D , Kamin-Lewis R , Cole G A
Published: January 2020

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.
Characterization and temporal regulation of mRNAs encoded by vaccinia virus intermediate-stage genes
Authors: Baldick C J , Moss B
Published: January 2020

The steady-state levels of mRNAs encoded by three intermediate-stage genes of vaccinia virus, A1L, A2L, and G8R, were compared with those encoded by well-characterized early- and late-stage genes. After synchronous infection of HeLa cells, the early mRNA was detected within 20 min and peaked at about 100 min; all three intermediate mRNAs were detected at 100 min and peaked at about 120 min; and the late mRNA was detected at 140 min and increased thereafter. Upon reaching maximum levels, the early and intermediate mRNAs declined at rates consistent with half-lives of about 30 min, providing the basis for rapid changes in gene expression. Intermediate mRNA was not detected when viral DNA synthesis was prevented, whereas its accumulation was enhanced by blocking translation after removal of the replication inhibitor. The 5' ends of the mRNAs initiated within a TAAAT or TAAAAT sequence in the coding DNA strand but contained a poly(A) leader of up to 30 additional bases. Diffuse bands of A1L and G8R RNA, equal to and longer than the coding region, were resolved by agarose gel electrophoresis, suggesting preferred sites of 3'-end formation that did not correlate with early gene termination signals. The cis-regulatory sequences were investigated by constructing recombinant viruses containing mutated intermediate promoters preceding the beta-galactosidase reporter gene. The effects of mutations on expression were similar to those previously obtained by transfection studies (C.J. Baldick, Jr., J.G. Keck, and B. Moss, J. Virol. 66:4710-4719, 1992), providing further evidence for functional core, spacer, and initiator regions. In addition, an up-regulated bifunctional early/intermediate promoter was created by making four single-base substitutions in the G8R promoter.
RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein
Authors: Riley D , Flint S J
Published: January 2020

The adenovirus L4 100-kDa nonstructural protein (100K protein) is required for efficient initiation of translation of viral late mRNA species during the late mRNA species during the late phase of infection (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). The RNA-binding properties of this protein were analyzed in an immunoprecipitation assay with the 100K-specific monoclonal antibody 2100K-1 (C. L. Cepko and P. A. Sharp, Virology 129:137-154, 1983). Coprecipitation of the 100K protein and 3H-infected cell RNA was demonstrated. The RNA-binding activity of the 100K protein was inhibited by single-stranded DNA but not by double-stranded DNA, double-stranded RNA, or tRNA. Competition assays were used to investigate the specificity with which the 100K protein binds to RNA in vitro. Although the protein exhibited a strong preference for the ribohomopolymer poly(U) or poly(G), no specific binding to viral mRNA species could be detected; uninfected or adenovirus type 5-infected HeLa cell poly(A)-containing and poly(A)-lacking RNAs were all effective inhibitors of binding of the protein to viral late mRNA. Similar results were obtained when the binding of the 100K protein to a single, in vitro-synthesized L2 mRNA was assessed. The poly(U)-binding activity of the 100K protein was used to compare the RNA-binding properties of the 100K protein prepared from cells infected by adenovirus type 5 and the H5ts1 mutant (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). A temperature-dependent decrease in H5ts1 100K protein binding was observed, correlating with the impaired translational function of this protein in vivo. By contrast, wild-type 100K protein RNA binding was unaffected by temperature. These data suggest that the 100K protein acts to increase the translational efficiency of viral late mRNA species by a mechanism that involves binding to RNA.
Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection
Authors: Tikhonenko A T , Linial M L
Published: January 2020

The avian retrovirus FH3, which encodes a Gag-Myc fusion protein, transforms chicken macrophages but not fibroblasts. However, passage of FH3 viral stock in fibroblasts leads to emergence of a virus capable of fibroblast transformation. This virus has not acquired myc mutations; instead, it carries internal gag deletions which confer the ability to transform fibroblasts. We now demonstrate that this and similar deletion variants emerge repeatedly during selection. Sequence analysis reveals direct repeats at or near deletion junctions, suggesting that errors during reverse transcription may be involved in genesis of these viruses, which are then positively selected in fibroblast culture. By using the polymerase chain reaction, we found that such variants preexisted in original stocks even before selection, although they could not be detected by focus assay.
In vitro transcription from baculovirus late gene promoters: accurate mRNA initiation by nuclear extracts prepared from infected Spodoptera frugiperda cells
Authors: Glocker B , Hoopes R R , Hodges L , Rohrmann G F
Published: January 2020

Extracts prepared from nuclei of Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells were shown to support in vitro transcription from baculovirus late gene promoters. In vitro transcription was optimized for the late promoter of the 39K gene. The Mg2+ concentration was critical; concentrations higher than 1 to 2 mM did not support late transcription. Additional conditions included template (40 micrograms/ml), extract (2.5 mg/ml), and incubation time (25 min). Using a combination of runoff assays and high-resolution primer extension analyses, this system was shown to accurately initiate transcription from a variety of baculovirus late gene promoters, including those from the 39K and p39/capsid late genes and the hyperexpressed p10 and polyhedrin very late genes. In vitro transcription from the 39K late promoter was resistant to high concentrations of both alpha-amanitin (100 micrograms/ml) and tagetitoxin (4,000 U/ml), suggesting that neither RNA polymerase II nor III is responsible for the transcription of baculovirus late genes.
Characterization of a naturally occurring ecotropic receptor that does not facilitate entry of all ecotropic murine retroviruses
Authors: Eiden M V , Farrell K , Warsowe J , Mahan L C , Wilson C A
Published: January 2020

A fibroblast cell line (MDTF) derived from the feral mouse Mus dunni is resistant to infection by Moloney murine leukemia virus (Mo-MuLV), an ecotropic murine leukemia virus (E-MuLV) (M. R. Lander and S. K. Chattopadadhyay, J. Virol. 52:695-698, 1984). MDTF cells can be infected by other E-MuLVs such as Friend MuLV and Rauscher MuLV, which have been demonstrated to use the same receptor as Mo-MuLV in NIH 3T3 cells (A. Rein and A. Schultz, Virology 136:144-152, 1984). We have now shown that the block to Mo-MuLV infection of MDTF cells occurs at the level of the envelope-receptor interaction. We have cloned the ecotropic receptor cDNA from MDTF cells (dRec) and compared its sequence with that of the NIH 3T3 cell receptor (mRec). Although the deduced dRec and mRec proteins differ at only four amino acid residues, we demonstrate that these changes account for the resistance of MDTF cells to Mo-MuLV infection. Our findings suggest that retroviruses in the same receptor class can exhibit different host ranges due to single amino acid differences in their cellular receptor.