[5] Characterization of coronavirus RNA polymerase gene products

Herold, Jens; Siddell, Stuart G.; Ziebuhr, John

doi:10.1016/s0076-6879(96)75007-3

Cited by 21 publications

(25 citation statements)

References 47 publications

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“…Upon induction, the temperature was shifted to 24°C. The cells were harvested after growing for another 4 h and 30 min, and the cell paste was suspended in column buffer (20 mM Tris-Cl [pH 8.0], 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol) and disrupted by sonication as described previously (22). Tween 20 (0.1%) was added, and the solution was centrifuged (20,000 ϫ g, 30 min) to produce a clear supernatant that was then loaded onto a column packed with amylose resin (New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Seybert

Dinten

Snijder

et al. 2000

J Virol

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The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5 single-stranded regions on the partial-duplex substrates, indicating a 5-to-3 polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.Equine arteritis virus (EAV) is the prototype of the Arteriviridae, a family of positive-stranded, enveloped RNA viruses which also includes Lactate dehydrogenase-elevating virus, Porcine reproductive and respiratory syndrome virus, and Simian haemorrhagic fever virus (for a review, see 47). A common ancestry of the Arteriviridae and Coronaviridae seems probable (6), and, consequently, the two families have been united in the order Nidovirales (3). The phylogenetic relationship between arteri-and coronaviruses is most evident from the organization and expression of their replicase genes. Thus, for example, both arteri-and coronaviruses (i) encode a very similar array of functional domains in their replicase genes, (ii) use ribosomal frameshifting to express key replicative functions, (iii) control the activity of the individual subunits of the viral replication and transcription machinery by extensive proteolytic processing of large protein precursors, and (iv) use a discontinuous transcription mechanism to produce a nested set of subgenomic (sg) mRNAs for structural gene expression (3,8).The EAV replicase gene comprises the 5Ј-terminal threefourths of the 12.7-kb genome and is composed of two open reading frames (ORFs), ORF1a and ORF1b (6). The upstream ORF1a encodes the ORF1a protein (187 kDa), and ORF1a and ORF1b together encode the ORF1ab protein (345 kDa). Expression of the ORF1b-encoded part of the ORFlab protein involves a ribosomal frameshift in the ORF1a-1b overlap region during translation of the genomic RNA (6). The primary translation products, which are also called replicase polyproteins, are extensively processed by three virus-encoded proteinases to produce 12 mature proteins (nonstructural protein 1 [nsp1] to nsp12), as well as multiple processing intermediates (for a recent review, s...

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Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Seybert

Dinten

Snijder

et al. 2000

J Virol

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“…The four predicted ORF1b nonstructural polypeptides comprise a putative RNA-dependent RNA polymerase (POL or nsp9), a protein (nsp10) containing a putative metal-binding domain (MBD) and nucleoside triphos-phate binding or helicase motif (HEL), and two proteins of unknown function (nsp 11 and 12). Nsp11 contains a domain (CORONA) that is conserved in all nidoviruses (Allende et al, 1999(Allende et al, , 2000den Boon et al, 1991;Godeny et al, 1993;Gorbalenya et al, 1989;Herold et al, 1996;Meulenberg et al, 1993;Nelsen et al, 1999;Shen et al, 2000;Snijder and Meulenberg, 1998;van Dinten et al, 1996Wootton et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…This information is necessary to devise effective strategies to prevent and control PRRSV infection of swine. The knowledge available for the genes encoded in ORF1ab is limited to sequence information for a few virus isolates (Allende et al, 1999(Allende et al, , 2000Jiang et al, 2000;Meulenberg et al, 1993;Nelsen et al, 1999;Shen et al, 2000;Wootton et al, 2000) and predictions on their function based on sequence homology and studies on EAV and coronaviruses (Gorbalenya et al, 1989;Herold et al, 1996;Snijder and Meulenberg, 1998;. To elucidate the role of the nonstructural genes of arteriviruses in virus replication, previous studies have been done with EAV (12.7 kb) to develop and characterize infectious clones and their derivative mutants (de Vries et al, 2000;van Dinten et al, 1997van Dinten et al, , 2000van Marle, 1999).…”

Section: Introductionmentioning

confidence: 99%

Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus

et al. 2002

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Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. One region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. PRRSV nsp10 was found to possess a thermolabile and pH-sensitive NTPase activity that was modulated by polynucleotides and to unwind dsRNA in a 5' to 3' polarity. These results provide the first evidence of the functional properties of PRRSV helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases.

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“…Plasmid DNAs were linearized with BamHI and transcripts were made with T7 RNA polymerase as described by Herold et al [13]. The synthetic cap structure m 7 G(5 )ppp(5 )G (Pharmacia) was included in the transcription reaction when the synthetic RNA was used for in vitro translations [7].…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

Characterization of an internal ribosome entry site within mRNA 5 of murine hepatitis virus

1999

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The unique region of mRNA 5 of murine hepatitis virus contains two open reading frames, ORF 5a and ORF 5b. The downstream ORF 5b encodes the envelope (E) protein, an integral membrane protein of the virus. We have shown previously that the expression of ORF 5b is mediated by the internal entry of ribosomes. In the experiments reported here, we have used the in vitro translation of synthetic mRNAs to identify the region of mRNA 5 that mediates internal ribosome entry. Our results show that the 5' border of the MHV mRNA 5 IRES element is located between nucleotides 227 and 244 in ORF 5a, while the 3' border is located between nucleotides 140 and 172 in ORF 5b. The MHV mRNA 5 IRES element, therefore, contains not more than 280 nucleotides and encompasses the ORF 5b initiation codon. As evidenced by electrophoretic mobility shift assays, the IRES element of mRNA 5 interacts specifically with protein factors present in an L-cell lysate.

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[5] Characterization of coronavirus RNA polymerase gene products

Cited by 21 publications

References 47 publications

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus

Characterization of an internal ribosome entry site within mRNA 5 of murine hepatitis virus

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