Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
J Virol ; 95(21): e0093621, 2021 10 13.
Article in English | MEDLINE | ID: mdl-34406860

ABSTRACT

An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate an REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus. We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, conversely, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also to understanding of the molecular regulation of orthobornavirus polymerase activity. IMPORTANCE Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus, is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus, can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.


Subject(s)
Borna disease virus/enzymology , Borna disease virus/metabolism , Genetic Vectors/metabolism , Nucleoproteins/metabolism , RNA-Dependent RNA Polymerase/metabolism , Viruses, Unclassified/metabolism , Amino Acid Sequence , Animals , Borna Disease/virology , Cell Nucleus/virology , Chlorocebus aethiops , HEK293 Cells , Humans , Plasmids/metabolism , RNA, Viral/metabolism , Reverse Genetics/methods , Vero Cells , Viral Proteins/metabolism , Virus Replication
2.
RNA ; 27(1): 54-65, 2021 01.
Article in English | MEDLINE | ID: mdl-33004436

ABSTRACT

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.


Subject(s)
Flaviviridae/ultrastructure , Host-Pathogen Interactions/genetics , RNA Folding , RNA, Untranslated/chemistry , RNA, Viral/chemistry , 3' Untranslated Regions , Animals , Base Pairing , Base Sequence , Cations, Divalent , Crystallography, X-Ray , Encephalitis Virus, Murray Valley/genetics , Encephalitis Virus, Murray Valley/metabolism , Encephalitis Virus, Murray Valley/ultrastructure , Exoribonucleases/chemistry , Exoribonucleases/metabolism , Flaviviridae/genetics , Flaviviridae/metabolism , Magnesium/chemistry , Magnesium/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Viruses, Unclassified/genetics , Viruses, Unclassified/metabolism , Viruses, Unclassified/ultrastructure , Zika Virus/genetics , Zika Virus/metabolism , Zika Virus/ultrastructure
3.
Nucleic Acids Res ; 44(5): 2362-77, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26783202

ABSTRACT

Halastavi árva virus (HalV) has a positive-sense RNA genome, with an 827 nt-long 5' UTR and an intergenic region separating two open reading frames. Whereas the encoded proteins are most homologous to Dicistrovirus polyproteins, its 5' UTR is distinct. Here, we report that the HalV 5' UTR comprises small stem-loop domains separated by long single-stranded areas and a large A-rich unstructured region surrounding the initiation codon AUG828, and possesses cross-kingdom internal ribosome entry site (IRES) activity. In contrast to most viral IRESs, it does not depend on structural integrity and specific interaction of a structured element with a translational component, and is instead determined by the unstructured region flanking AUG828. eIF2, eIF3, eIF1 and eIF1A promote efficient 48S initiation complex formation at AUG828, which is reduced ∼5-fold on omission of eIF1 and eIF1A. Initiation involves direct attachment of 43S preinitiation complexes within a short window at or immediately downstream of AUG828. 40S and eIF3 are sufficient for initial binding. After attachment, 43S complexes undergo retrograde scanning, strongly dependent on eIF1 and eIF1A. eIF4A/eIF4G stimulated initiation only at low temperatures or on mutants, in which areas surrounding AUG828 had been replaced by heterologous sequences. However, they strongly promoted initiation at AUG872, yielding a proline-rich oligopeptide.


Subject(s)
Genome, Viral , Peptide Chain Initiation, Translational , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , Ribosomes/metabolism , Viral Proteins/biosynthesis , Viruses, Unclassified/metabolism , 5' Untranslated Regions , Animals , Cell-Free System , Cloning, Molecular , Codon, Initiator/chemistry , Codon, Initiator/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Gene Expression , Nucleic Acid Conformation , Open Reading Frames , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reticulocytes/chemistry , Ribosomes/chemistry , Spodoptera/chemistry , Viral Proteins/genetics , Viruses, Unclassified/genetics
4.
J Virol ; 51(1): 7-13, 1984 Jul.
Article in English | MEDLINE | ID: mdl-6202887

ABSTRACT

The polypeptides of the highly virulent mink-passaged Utah I and the nonvirulent cell culture-adapted ADV-G strain of Aleutian disease virus (ADV) were compared. When CRFK cells infected with either Utah I or ADV-G were analyzed by immunoprecipitation, both viruses induced proteins with molecular weights characteristic of the ADV-G 85,000 ( 85k )- and 75k-dalton structural proteins (p85 and p75) as well as the 71k -dalton nonvirion protein p71 . However, when Utah I, Pullman ADV, and DK ADV (a Danish isolate of ADV) were purified from infected mink, only polypeptides with molecular weights between 27k and 30k could be identified. In addition, trypsin treatment of ADV-G degraded p85 and p75 to smaller antigenic proteins with molecular weights of 24k and 27k, similar to those found for the virulent in vivo viruses. The effect of proteolytic treatment of ADV was then studied in detail. Purification of Utah I ADV from mink organs in the presence of protease inhibitor did not prevent the appearance of the low-molecular-weight proteins and ADV-G proteins were not degraded upon purification from a homogenate of normal mink organs, suggesting that artifactual proteolysis was not occurring. When a serum pool from terminally diseased mink was analyzed by radioimmunoassay for antibody reactivity against trypsinized and nontrypsinized ADV-G, five times higher reactivity was found for the trypsinized ADV-G than for the nontrypsinized ADV-G, an effect which could not be elicited by chymotrypsin or V8 protease treatment, implying that in vivo-produced ADV was being modulated in vivo by trypsin or a trypsin-like enzyme. Trypsinization was shown not to cause a change in ADV virion density, but to decrease the in vitro infectivity of ADV-G for CRFK cells. These studies suggested that during infection of mink ADV proteins are degraded to highly antigenic smaller polypeptides.


Subject(s)
Aleutian Mink Disease Virus/metabolism , Aleutian Mink Disease/microbiology , Viruses, Unclassified/metabolism , Aleutian Mink Disease Virus/immunology , Animals , Centrifugation, Isopycnic , Epitopes/analysis , Mink , Molecular Weight , Peptide Hydrolases/metabolism , Viral Proteins/analysis , Virion/analysis
6.
J Virol ; 10(2): 228-33, 1972 Aug.
Article in English | MEDLINE | ID: mdl-4116344

ABSTRACT

Maedi virus contains a ribonucleic acid (RNA) which can be resolved into three major components, namely, 62S, 33S, and 13S, by sucrose gradient centrifugation. The presence of RNA- and deoxyribonucleic acid (DNA)-dependent DNA polymerase in virions of maedi virus was demonstrated. The enzyme product could be converted into acid-soluble form by pancreatic deoxyribonuclease, but was resistant to digestion by pancreatic ribonuclease and to hydrolysis by NaOH.


Subject(s)
RNA, Viral/analysis , RNA-Directed DNA Polymerase/analysis , Viruses, Unclassified/analysis , Animals , Centrifugation, Density Gradient , Choroid Plexus , Culture Techniques , DNA, Viral/biosynthesis , Hydrolysis , Pulmonary Adenomatosis, Ovine/microbiology , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/metabolism , Ribonucleases , Sheep , Sodium Hydroxide , Thymidine/metabolism , Thymine Nucleotides/metabolism , Tritium , Uridine/metabolism , Viruses, Unclassified/enzymology , Viruses, Unclassified/growth & development , Viruses, Unclassified/metabolism
8.
J Virol ; 8(4): 573-8, 1971 Oct.
Article in English | MEDLINE | ID: mdl-4108574

ABSTRACT

The physical and biochemical characteristics of progressive pneumonia virus were found to be remarkably similar to those of the ribonucleic acid (RNA) tumor viruses. Significant findings included the presence of a 60 to 70S RNA genome, RNA-dependent deoxyribonucleic acid polymerase activity, and common morphological properties. This information correlates with previously reported biological observations and supports the provisional inclusion of enveloped RNA-containing "slow viruses" within the RNA tumor virus group.


Subject(s)
Viruses, Unclassified , Animals , Cell Line , Centrifugation, Density Gradient , Chemical Precipitation , DNA Nucleotidyltransferases/metabolism , Deoxyribonucleases , Ethanol , Hydrolysis , Male , Microscopy, Electron , Phosphates/metabolism , Phosphorus Isotopes , Pulmonary Adenomatosis, Ovine , RNA, Viral/isolation & purification , Ribonucleases , Sheep , Staining and Labeling , Sucrose , Testis , Thymidine/metabolism , Tritium , Uridine/metabolism , Virus Cultivation , Viruses, Unclassified/classification , Viruses, Unclassified/enzymology , Viruses, Unclassified/isolation & purification , Viruses, Unclassified/metabolism
9.
J Virol ; 7(6): 726-35, 1971 Jun.
Article in English | MEDLINE | ID: mdl-4327891

ABSTRACT

A ribonucleic acid (RNA)-dependent RNA polymerase has been demonstrated in Kern Canyon virus (KCV) particles. The RNA product of the KCV polymerase hybridizes to KCV viral RNA. The properties of this viral enzyme have been characterized and compared with those of vesicular stomatitis virus (VSV). RNA polymerases from both viruses require similar conditions of temperature, pH, and detergent and magnesium concentrations for maximal synthesis of RNA. The RNA polymerase contained in the virion of KCV was more dependent on the presence of a sulfhydryl agent than was the VSV enzyme. Under optimal conditions, the specific activity of the VSV polymerase is about twenty-five times as great as that of KCV.


Subject(s)
RNA Nucleotidyltransferases/metabolism , Vesicular stomatitis Indiana virus/enzymology , Viruses, Unclassified/enzymology , Animals , Carbon Isotopes , Cell Line , Centrifugation, Density Gradient , Chemical Precipitation , Cricetinae , Culture Media , Guanine Nucleotides/metabolism , Hydrogen-Ion Concentration , Kidney , Magnesium/pharmacology , Nucleic Acid Hybridization , Quaternary Ammonium Compounds , RNA Nucleotidyltransferases/isolation & purification , RNA, Viral/biosynthesis , Species Specificity , Sucrose , Sulfates , Sulfhydryl Reagents/pharmacology , Temperature , Tritium , Uracil Nucleotides/metabolism , Uridine/metabolism , Vesicular stomatitis Indiana virus/isolation & purification , Vesicular stomatitis Indiana virus/metabolism , Vesicular stomatitis Indiana virus/pathogenicity , Viruses, Unclassified/isolation & purification , Viruses, Unclassified/metabolism , Viruses, Unclassified/pathogenicity
SELECTION OF CITATIONS
SEARCH DETAIL
...