bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication
Authors: Chambers T J , Nestorowicz A , Amberg S M , Rice C M
Published: January 2020

To study the role of specific regions of the yellow fever virus NS2B protein in proteolytic processing and association with the NS3 proteinase domain, a series of mutations were created in the hydrophobic regions and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cleavage at the 2B/3 cleavage site and on processing at other consensus cleavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression in BHK cells. Association between NS2B and the NS3 proteinase domain and the effects of mutations on complex formation were investigated by nondenaturing immunoprecipitation of these proteins expressed in infected cells, by cell-free translation, or by recombinant vaccinia viruses. Mutations within the hydrophobic regions had subtle effects on proteolytic processing, whereas mutations within the conserved domain dramatically reduced cleavage efficiency or abolished all cleavages. The conserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eliminate both association of these two proteins and trans-cleavage activity. In addition, mutations which either eliminated proteolytic processing or had no apparent effect on processing were found to abolish recovery of infectious virus following RNA transfection. These results suggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose structural integrity may not be essential for proteolytic processing may have additional functions during viral replication.
Anti-idiotypic mimicry of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus
Authors: Tackaberry E S , Hamel J , Larose Y , Brodeur B R
Published: January 2020

Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.
Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus
Authors: Love D N , Bell C W , Pye D , Edwards S , Hayden M , Lawrence G L , Boyle D , Pye T , Whalley J M
Published: January 2020

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.
Occurrence of alternatively spliced leader-delta onc-poly(A) transcripts in chicken neuroretina cells infected with Rous-associated virus type 1: implication in transduction of the c-mil/c-raf and c-Rmil/B-raf oncogenes
Authors: Felder M P , Laugier D , Eychene A , Calothy G , Marx M
Published: January 2020

We previously reported that serial passaging of Rous-associated virus type 1 in nondividing chicken embryo neuroretina cells leads to reproducible generation of acutely mitogenic retroviruses that transduced the catalytic domain of c-mil/c-raf or c-Rmil/B-raf. On the basis of structural analysis of several retroviruses, we proposed that the early step of oncogene transduction is the constitution of alternatively spliced leader-delta onc-poly(A) transcripts. Here, we show that neuroretina cells do synthesize hybrid leader-delta mil and leader-delta Rmil RNAs and that these RNAs exhibit mitogenic properties and serve as templates for the generation of transducing retorviruses.
Generation of a water-soluble oligomeric ectodomain of the Rous sarcoma virus envelope glycoprotein
Authors: Gilbert J M , Hernandez L D , Chernov-Rogan T , White J M
Published: January 2020

Sequences encoding the transmembrane domain of the Rous sarcoma virus envelope (Env) glycoprotein were deleted and replaced with sequences that signal addition of a glycosyl phosphatidylinositol (GPI) membrane anchor. Stable NIH 3T3 cell lines expressing either the wild-type transmembrane-anchored Env or the Env chimera with a GPI tail were established. The GPI-anchored envelope glycoprotein is expressed, oligomerized, and transported to the cell surface in a manner identical to that of its wild-type transmembrane-anchored counterpart. The GPI-linked protein is quantitatively removed from the cell surface by treatment with phosphatidylinositol phospholipase C. The phosphatidylinositol phospholipase C-released, water-soluble Env glycoprotein ectodomain retains the wild-type oligomeric structure and provides a useful tool for studying the subgroup-specific binding and fusion activities of a prototypic retroviral Env glycoprotein.
Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication
Authors: Pari G S , Anders D G
Published: January 2020

Recently we described the use of human cytomegalovirus (HCMV) cosmid clones in a cotransfection assay of HCMV oriLyt replication (G. S. Pari, M. A. Kacica, and D. G. Anders, J. Virol. 67:2575-2582, 1993). We have now used this assay to identify 11 distinct required loci encoding trans-acting factors sufficient for transient complementation of oriLyt-dependent DNA replication. This set includes all of the virus genes essential to initiate and perform DNA synthesis together with the virus genes required to express these replication functions from their native promoters. Six of the identified loci span open reading frames (ORFs) that encode homologs or probable homologs of herpes simplex virus type 1 replication genes, consistent with predictions based on sequence similarities and biochemical properties. These include the DNA polymerase UL54 and polymerase-associated protein UL44, the single-stranded-DNA-binding protein UL57, and proposed subunits of a helicase-primase complex, UL70, UL105, and UL101-102. Frameshift mutations in any one of these essential ORFs abrogated complementation of DNA replication. Three required loci, UL36-38, IRS1 (or TRS1), and IE1/IE2, encode known regulatory proteins. The remaining two loci span ORFs UL84 and UL112-113 and encode early temporal class nucleus-associated proteins of unknown function. Neither of these genes have been implicated previously in DNA replication or in regulating gene expression, nor have counterparts in herpes simplex virus type 1 or Epstein-Barr virus been described. The results presented here will facilitate investigation of the mechanisms and regulation of HCMV lytic-phase DNA replication.
Human T-cell leukemia virus type 1 Tax activates transcription of the human fra-1 gene through multiple cis elements responsive to transmembrane signals
Authors: Tsuchiya H , Fujii M , Niki T , Tokuhara M , Matsui M , Seiki M
Published: January 2020

We have shown that Tax1 of human T-cell leukemia virus type 1 stimulates the expression of several cellular immediate-early genes (M. Fujii, T. Niki, T. Mori, T. Matsuda, M. Matsui, N. Nomura, and M. Seiki, Oncogene 6:1023-1029, 1991). In this study, the 5'-flanking region of the human fra-1 gene, which is a Tax1-inducible fos-related gene, was isolated and Tax1 or serum-responsive cis elements were analyzed to obtain further insight into the mechanism of Tax1 action. The 62-bp sequence starting 46 nucleotides upstream from the translation initiation site showed 71% homology with the sequence surrounding the TATA box of the c-fos promoter. Regulatory motifs identified in the c-fos promoter, such as an Ets-binding site, E boxes, a CArG box, c-fos AP-1 sites, and two retinoblastoma control elements, were also found upstream of the c-fos homology region. A 502-bp fragment containing these motifs mediated transcriptional activation by Tax1 or by serum in a transient transfection assay. Three independent Tax1-responsive regions (TRRs) were identified, and mutations in each revealed that one of the retinoblastoma control elements in TRR1 and the c-fos AP-1 sites in TRR2 and TRR3 were essential for the activation. Although TRR2 contains a CArG box-like sequence, it was a weak binding site for p67SRF, if it bound at all, and was not required for activation. All three TRRs could also mediate the signals stimulated by serum. Thus, Tax1 appears to activate fra-1 gene expression by means of a part of the cellular machinery similar to that which mediates growth signals.
Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain
Authors: Stohlman S A , Kyuwa S , Polo J M , Brady D , Lai M M , Bergmann C C
Published: January 2020

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.
Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus
Authors: Stark R , Meyers G , Rümenapf T , Thiel H J
Published: January 2020

The polyprotein of classical swine fever virus starts with the nonstructural protein p23, which is followed by the nucleocapsid protein p14. Proteolytic cleavage between p23 and p14 was demonstrated in a cell-free transcription-translation system. Successive truncation of the cDNA used for the transcription indicated that the proteolytic activity responsible for the cleavage between p23 and p14 resides within p23. In order to determine the cleavage site between these two proteins, the respective genomic regions were expressed in two different expression systems. N-terminal sequencing of the resulting p14-related proteins revealed that cleavage occurs between Cys-168 and Ser-169. Comparison of the sequence around the cleavage site with sequences of other pestiviruses suggests a conserved processing site between similar proteins.
Two E2 binding sites (E2BS) alone or one E2BS plus an A/T-rich region are minimal requirements for the replication of the human papillomavirus type 11 origin
Authors: Lu J Z , Sun Y N , Rose R C , Bonnez W , McCance D J
Published: January 2020

Human papillomaviruses (HPVs) cannot be propagated in vitro, but the DNA can be replicated transiently in an assay in the presence of two trans-acting viral proteins, E1 and E2. Using this assay, we have defined the minimal cis-acting elements of the origin of replication of HPV type 11. Most HPV genomes are conserved at the origin of replication, and the core contains three E2 binding sites (E2BS) surrounding an A/T-rich spacer region. The present results show that the minimal requirement for replication is either two E2BS alone or the A/T-rich region plus one E2BS; in the latter case the relative position of the E2BS is important. In all the studies, the presence of both E1 and E2 proteins was essential for replication, yet only the E2BS was required at the origin. We have shown that E1, E2, and the origin of replication containing an E2BS from a complex in vitro, and our data are consistent with a model in which E2 acts to target E1 to the HPV type 11 replication origin.
Mutually exclusive binding of two cellular factors within a critical promoter region of the gene for the IE110k protein of herpes simplex virus
Authors: O'Rourke D , O'Hare P
Published: January 2020

We have examined the cis- and trans-acting factors involved in constitutive transcription of the promoter for the IE110k protein of herpes simplex virus type 1. Our results indicate that while the IE110k gene is activated by Vmw65, it also exhibits very efficient constitutive expression approximating that from the simian virus 40 early enhancer-promoter region. We show that despite the presence of multiple copies of the octamer consensus site which mediate Oct-1 binding and subsequent Vmw65 activation, these upstream sequences have a minor effect on constitutive transcription. By progressive exonuclease digestion and subsequent site-directed mutagenesis of the promoter, we have identified a 15-bp region (termed the EC region), from position -89 to -74, which is required for efficient constitutive expression from the IE110k promoter. We demonstrate that two cellular proteins interact with this region and, by competition and methylation interference analyses, show they have distinct but overlapping sequence requirements for binding. One of these proteins is identified as NF-Y, a CCAAT box-binding factor, which binds an inverted CCAAT box located between positions -71 and -75. The second cellular factor, F2, appears to be novel and binds a region with the sequence CGCGCGGC CAT which overlaps the 3' end of the CCAAT box. The terminal AT of the recognition site for F2 represents, on the opposite strand, the terminal AT of the CCAAT box, and these and adjacent bases are critically required for the binding of both factors. These results together with further competition analysis indicate that these factors bind in a mutually exclusive manner to the EC region. The implications of these results for regulation of expression of the IE110k gene are discussed.
Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation
Authors: Ansardi D C , Morrow C D
Published: January 2020

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification of the activation domain of equine infectious anemia virus rev
Authors: Fridell R A , Partin K M , Carpenter S , Cullen B R
Published: January 2020

Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have identified a segment of this EIAV protein that can efficiently substitute in cis for the otherwise essential activation motif. Interestingly, the minimal EIAV Rev activation motif identified in this study comprises approximately 18 amino acids located toward the protein N terminus that lack any evident similarity to the leucine-rich activation domains found in these other lentivirus Rev proteins. It therefore appears that the Rev protein of EIAV, while analogous in function to Rev proteins defined in lentiviruses of primate, ovine, and caprine origin, is nevertheless distinguished by an entirely novel domain organization.
Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus
Authors: Matloubian M , Kolhekar S R , Somasundaram T , Ahmed R
Published: January 2020

This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K-->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-->K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K-->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F-->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.
Site-specific inhibition of RNA polymerase II preinitiation complex assembly by human cytomegalovirus IE86 protein
Authors: Wu J , Jupp R , Stenberg R M , Nelson J A , Ghazal P
Published: January 2020

The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein (UL122, IE2), is a DNA-binding protein that represses the MIEP through its cognate recognition sequence (designated the cis repression signal [crs]) located between the TATA box and the initiation site of transcription. Purified recombinant IE86 protein was shown to repress MIEP transcription in vitro, in a cis-acting mediated pathway, with nuclear extracts from HeLa S3, U373-MG, and primary human foreskin fibroblast cells. Repression of the MIEP by IE86 was shown by two criteria to be dependent on the direct interaction of IE86 with the crs element. Core promoter constructs containing essentially the MIEP TATA box and crs element were also specifically repressed by IE86 but not by a mutant IE86 protein, indicating the general transcription machinery as the target for IE86 repression. Kinetic and template commitment experiments demonstrated that IE86 affects preinitiation complex formation but not the rate of reinitiation. Sarkosyl inhibition experiments further revealed that IE86 was unable to effect repression by either disassembling or preventing the elongation of a preexisting transcription complex. Further, the ability of IE86 to interact with the DNA-binding subunit of TFIID was shown not to be required for repression. These functional protein-DNA and protein-protein interaction experiments demonstrate that IE86 specifically interferes with the assembly of RNA polymerase II preinitiation complexes. The biological significance of these results and the precise mechanism by which IE86 represses transcription are discussed.
NF-kappa B p100 is one of the high-molecular-weight proteins complexed with the v-Rel oncoprotein in transformed chicken spleen cells
Authors: Sif S , Gilmore T D
Published: January 2020

The Rel/NF-kappa B family of proteins includes several interacting cellular transcription factors and the v-Rel oncoprotein of the avian Rev-T retrovirus. We report the isolation of a chicken cDNA for the NF-kappa B p52 precursor protein p100. Full-length p100 only weakly binds DNA in vitro; removal of the ankyrin-like repeats generates C-terminally truncated p100 proteins (like p52) that have an increased ability to bind an oligonucleotide containing a kappa B site. In addition, we show that chicken p100 is identical to a protein previously designated p115, which is found in a complex with v-Rel in transformed chicken spleen cells. Furthermore, p100 and v-Rel can form a complex when synthesized in vitro. Using cDNAs for chicken NF-kappa B p105, NF-kappa B p100, c-Rel, and v-Rel, we show that one of the complexes in v-Rel-transformed spleen cells can be reconstituted in vitro.
Mutants of Epstein-Barr virus with a selective marker disrupting the TP gene transform B cells and replicate normally in culture
Authors: Kim O J , Yates J L
Published: January 2020

We have isolated mutants of Epstein-Barr virus (EBV) which carry a dominant selectable marker inserted into the third exon of the gene encoding two membrane proteins, TP1 and TP2 (or LMP2A and LMP2B), which are expressed in latently infected, growth-transformed B cells. One of the mutants also acquired a 260-bp deletion beginning in the first intron a few base pairs from the terminal repeats and removing most of the second TP exon, including the initial coding sequences of TP2. These EBV mutants transform human B cells in culture, and the transformed B-cell clones carrying them release EBV at approximately normal frequencies.
Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice: a laboratory model of natural selection
Authors: Golovkina T V , Prescott J A , Ross S R
Published: January 2020

Transgenic mice that expressed the superantigen protein encoded in the C3H exogenous mouse mammary tumor virus long terminal repeat deleted their V beta 14+ T cells during the shaping of their immune repertoire and showed no evidence of virus production in their mammary glands after infection by milk-borne C3H exogenous virus. However, they developed mammary gland tumors that had newly integrated copies of C3H exogenous virus, although the latency of tumor formation was much longer than in their nontransgenic littermates that retained their V beta 14+ T cells. After four generations, infectious C3H virus was completely eliminated from the transgenic mouse pedigree. These data support the hypothesis that endogenous mouse mammary tumor proviruses are retained in the genome as protection against exogenous virus infection and subsequent tumorigenesis and show that there may be natural selection against the virus in vivo.
Frequent polymerase errors observed in a restricted area of clones derived from the attachment (G) protein gene of respiratory syncytial virus
Authors: Cane P A , Matthews D A , Pringle C R
Published: January 2020

Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues. Such mutations were detected in 14% of clones derived from mRNA, 58% of clones derived from genomic-sense RNA, and 50% of clones derived from in vitro-transcribed RNA. This phenomenon appears to be dependent on the template sequence.
Induction of renal adenocarcinoma by a nonmutated erbB oncogene
Authors: Taglienti-Sian C A , Banner B , Davis R J , Robinson H L
Published: January 2020

Oncogenicity tests have revealed that a nonmutated erbB oncogene induces renal adenocarcinoma in addition to erythroblastosis. The erbB oncogene is a truncated form of the chicken epidermal growth factor receptor that lacks the extracellular ligand-binding domain. Previously, the nonmutated erbB oncogene has been reported to cause only erythroblastosis. The expansion of the disease potential of erbB to additional neoplasms has been associated with mutations (truncations, deletions, and point mutations) within the erbB gene. Our results indicate that a nonmutated virally expressed erbB oncogene (REB-c) causes a 100% incidence of renal neoplasia.
Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells
Authors: Caughey B , Raymond G J
Published: January 2020

The accumulation of an abnormal, protease-resistant form of the protein PrP (PrP-res) in hosts with scrapie and related transmissible spongiform encephalopathies appears to be important in disease pathogenesis. To gain insight into the mechanism of PrP-res accumulation and the in vivo antiscrapie activity of certain polyanions, we have studied effects of sulfated glycans on PrP metabolism in scrapie-infected neuroblastoma cells. Pentosan polysulfate, like the amyloid-binding dye Congo red, potently inhibited the accumulation of PrP-res in these cells without apparent effects on the metabolism of the normal isoform. The inhibition was due primarily to prevention of new PrP-res accumulation rather than destabilization of preexisting PrP-res. PrP-res accumulation remained depressed in the cultures after removal of the inhibitors. The activities of other sulfated glycans, nonsulfated polyanions, dextran, and DEAE-dextran were compared with those of pentosan polysulfate and Congo red. This comparison provided evidence that the density of sulfation and molecular size are factors influencing anti-PrP-res activity of sulfated glycans. The relative potencies of these compounds corresponded well with their previously determined antiscrapie activities in vivo, suggesting that the prophylactic effects of sulfated polyanions may be due to inhibition of PrP-res accumulation. Since PrP-res amyloid is known to contain sulfated glycosaminoglycans, we reason that these inhibitors may competitively block an interaction between PrP and endogenous glycosaminoglycans that is essential for its accumulation in a protease-resistant, potentially amyloidogenic state.
Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV
Authors: Tikoo S K , Parker M D , van den Hurk J V , Kowalski J , Zamb T J , Babiuk L A
Published: January 2020

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.
The DNA-binding domain of two bZIP transcription factors, the Epstein-Barr virus switch gene product EB1 and Jun, is a bipartite nuclear targeting sequence
Authors: Mikaélian I , Drouet E , Marechal V , Denoyel G , Nicolas J C , Sergeant A
Published: January 2020

The Epstein-Barr virus BZLF1 gene product EB1 (also called ZEBRA and Zta), is a transcription factor belonging to the bZIP (basic domain leucine zipper) family of nuclear proteins. Translocation to the nucleus of EB1 (J. Becker, U. Leser, M. Marschall, A. Langford, W. Jilg, H. Gelderblom, P. Reichart, and H. Wolf, Proc. Natl. Acad. Sci. USA 88:8332-8336, 1991) and of two other bZIP proteins, c-Jun and c-Fos (P. Roux, J.-M. Blanchard, A. Fernandez, N. Lamb, P. Jeanteur, and M. Piechaczyk, Cell 63:341-351, 1990), has been shown to be subject to regulation. We show here that for both EB1 and Jun the nuclear targeting signals (NTS) in the proteins' primary sequences are two clusters of positively charged amino acids. These clusters, called BRA and BRB, are necessary and sufficient to direct beta-galactosidase to the nuclear compartment and act as a bipartite NTS. They are conserved among all the bZIP proteins, and although they are not identical, they probably share the same function. Site-directed mutagenesis studies made on these basic clusters suggest that they also act as a bipartite NTS in the EB1 protein. Our results also demonstrate that in EB1 and Jun, these bipartite NTS are superimposed with bipartite DNA-binding domains, since BRA and BRB are required in vitro for direct and specific contact between these proteins and their DNA-binding sites.
RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria
Authors: Warrener P , Tamura J K , Collett M S
Published: January 2020

The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.
Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate
Authors: Trybala E , Svennerholm B , Bergström T , Olofsson S , Jeansson S , Goodman J L
Published: January 2020

We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction.
Cooperation of structural proteins during late events in the life cycle of polyomavirus
Authors: Forstová J , Krauzewicz N , Wallace S , Street A J , Dilworth S M , Beard S , Griffin B E
Published: January 2020

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.
Asymmetric resolution of a parvovirus palindrome in vitro
Authors: Cotmore S F , Nüesch J P , Tattersall P
Published: January 2020

Cell extracts from murine A9 or human HeLa cells containing wild-type copies the NS1 polypeptide of minute virus of mice (MVM), produced from a recombinant vaccinia virus, can support the resolution of viral 3' termini from palindromic junction fragments of dimeric, replicative-form MVM DNA. Resolution resulted in the generation of two new viral termini, one associated with each arm of the junction palindrome. Telomeres were created in two configurations, "extended" forms, which were covalently associated with NS1 molecules, and smaller "turn-around" forms in which a single arm of the palindrome terminated at the axis of dyad symmetry in a covalent bond which cross-linked the two strands. The in vitro resolution reaction was asymmetric, generating predominantly extended-form termini from one arm of the palindrome but predominantly turn-around forms from the other. This asymmetry was independent of the type of cell used to prepare the in vitro extract or the orientation of the palindrome in the plasmid and was obtained for all cloned junction sequences of 156 bp or more. Two modified forms of the duplex junction fragment, which appeared to be intermediates in the resolution process since they were nicked, covalently linked to NS1, and associated with newly synthesized DNA, were identified. The structures of these intermediates suggest that resolution is initiated by preferential nicking at one of the two candidate resolution sites. The asymmetric nature of this resolution reaction is discussed in terms of current models of MVM DNA replication.
Analysis of the human T-cell response to picornaviruses: identification of T-cell epitopes close to B-cell epitopes in poliovirus
Authors: Graham S , Wang E C , Jenkins O , Borysiewicz L K
Published: January 2020

Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.
Disulfide bond formation in the human immunodeficiency virus type 1 Nef protein
Authors: Zazopoulos E , Haseltine W A
Published: January 2020

Substitution of alanine for cysteine residues of the human immunodeficiency virus type 1 LAI (BRU) and ELI Nef proteins was used to determine pairing of the cysteine residues present in each protein. The results show that under nonreducing conditions, alternative pairing of the cysteines occurs. The preferred pairing of cysteine residues of the LAI and ELI proteins differs. In the experimental system used, viruses carrying the ELI nef allele are found to express Nef proteins which accelerate virus replication. Mutation in critical cysteine residues of the protein reduce the rate of virus replication. In the same system, viruses harboring the LAI nef allele fail to replicate. These observations raise the possibility that differences in the observed biological activity of nef alleles may be attributed, at least in part, to differences in the secondary structure of the proteins.
Papillomas and carcinomas in transgenic rabbits carrying EJ-ras DNA and cottontail rabbit papillomavirus DNA
Authors: Peng X , Olson R O , Christian C B , Lang C M , Kreider J W
Published: January 2020

Two transgenic rabbits (TRI and TRIII) that carried cottontail rabbit papillomavirus (CRPV) DNA alone were identified; another (TRII) carried both CRPV DNA and EJ-ras. TRI and TRIII developed extensive skin papillomas at about 1 month of age, and transcripts of CRPV DNA were detectable only in skin and/or papillomas. TRII developed extensive squamous carcinomas of the skin at a very early age. Transcription of both CRPV DNA and EJ-ras was found in the skin cancers. Thus, the tissue specificity of CRPV DNA expression in transgenic rabbits was the same as in virion-infected animals. The expression of EJ-ras could be dependent on the expression of certain CRPV genes and may be a critical cofactor of CRPV DNA in the progression of carcinomas.
Replication and persistence of simian immunodeficiency virus variants after passage in macaque lymphocytes and established human cell lines
Authors: Rudensey L M , Papenhausen M D , Overbaugh J
Published: January 2020

In lentivirus infections, there are typically few cells in the host that harbor the provirus. For this reason, molecular clones of human and simian immunodeficiency viruses (HIV and SIV) are generally derived after passage and amplification of the virus in cell culture. To determine whether SIV variants that persist in culture are similar to the variants that predominate in the host, we examined the proviral sequence of the SIV envelope (env) gene before and after cocultivation of lymphocytes from a macaque with AIDS with naive macaque lymphocytes or human cell lines. Many of the predominant variants in the monkey replicated and persisted in macaque lymphocytes and CEMx174 cells in culture, but a more limited population of variants replicated in C8166 cells. Passage of virus, harvested after 4 weeks of coculture, onto naive cells further demonstrated that the majority of proviruses detected by polymerase chain reaction were also viral variants that were expressed and packaged into infectious virions.
A binding site for transcription factor E2F is a target for trans activation of murine thymidine kinase by polyomavirus large T antigen and plays an important role in growth regulation of the gene
Authors: Ogris E , Rotheneder H , Mudrak I , Pichler A , Wintersberger E
Published: January 2020

The promoter of the murine thymidine kinase gene contains a binding site for transcription factor E2F. Using cell lines (3T3-LT) conditionally expressing polyomavirus large T antigen from a hormone-responsive promoter and reporter gene constructs carrying the thymidine kinase promoter with intact or mutated E2F sites, we show that this E2F site is the target for trans activation by the viral protein. Transcription of the growth-regulated endogenous thymidine kinase gene can be activated in serum-starved, quiescent 3T3-LT cells by induction of T antigen. Activation of transcription from the thymidine kinase promoter requires an intact binding site for the retinoblastoma protein in the T antigen. The same promoter region was furthermore shown to play a major role in growth regulation of the gene. As several other DNA synthesis enzymes also carry E2F binding sites in their promoters, our observations suggest a common mechanism of growth regulation of these genes and that they all might be targets for trans activation by DNA tumor virus proteins.