scholarly journals Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

2004 ◽  
Vol 78 (11) ◽  
pp. 5619-5632 ◽  
Author(s):  
Konstantin A. Ivanov ◽  
Volker Thiel ◽  
Jessika C. Dobbe ◽  
Yvonne van der Meer ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Enzymatic Activities ◽  
Membrane Structures ◽  
Processing Enzymes ◽  
Cytoplasmic Rna ◽  
Group 2 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

NUCLEIC ACID SYNTHESIS AND TRANSFER IN NORMAL, SECOND GENERATION CHICK EMBRYO FIBROBLASTS

1961 ◽  
Vol 39 (10) ◽  
pp. 1625-1633 ◽  
Author(s):  
R. Bather ◽  
Elizabeth Purdie-Pepper
Keyword(s):  
Chick Embryo ◽  
Rna Synthesis ◽  
Second Generation ◽  
Acid Synthesis ◽  
Quantitative Estimates ◽  
Cytoplasmic Rna ◽  
Embryo Fibroblasts ◽  
Slight Rise ◽  
Zero Time ◽  
Rna And Dna

The stripping film technique of autoradiography has been used to study some aspects of RNA and DNA metabolism in chick embryo fibroblasts in second generation tissue culture.Approximately one third of the cells incorporated thymidine-H3into DNA in a 20-minute uptake period. The duration of DNA synthesis, the generation time (time elapsing between two successive cell divisions), and the duration of mitosis have been calculated and are very similar to the values obtained for pure strains of hamster cells maintained in culture for several months by another author.RNA synthesis, as measured by uridine-H3incorporation, occurred only in the nucleus and nucleolus. Both sites synthesized RNA simultaneously beginning at zero time. The ratio of the number of grains over the nucleolus to that over the whole nucleus remained constant throughout the uptake of uridine-H3and its transfer to the cytoplasm.A small amount of labelled soluble RNA precursors remain in the nucleus after removal of uridine-H3from the medium. This results in a slight rise in radioactivity of the nucleus after uridine-H3removal. RNA then leaves the nucleus rapidly and appears in the cytoplasm. The half life of RNA in the nucleus is about 4 hours. Some turnover of cytoplasmic RNA seems to occur after 8 hours but quantitative estimates of its rate cannot be made due to changing geometry of the cells as the RNA migrates from the nuclear to the peripheral parts of the cell.Finally, little or no RNA synthesis occurs for a period of about 30 minutes during contraction of the chromosomes in mitosis.

scholarly journals Intracellular Distribution of Rubella Virus Nonstructural Protein P150

1999 ◽  
Vol 73 (9) ◽  
pp. 7805-7811 ◽  
Author(s):  
Pekka Kujala ◽  
Tero Ahola ◽  
Neda Ehsani ◽  
Petri Auvinen ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Rubella Virus ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Vero Cells ◽  
Intracellular Distribution ◽  
Viral Rna ◽  
Membrane Structures ◽  
Tubular Membrane ◽  
Amino Terminal ◽  
Amino Terminal Fragment

ABSTRACT Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells. Replicase protein P150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. Newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral RNA synthesis.

scholarly journals A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases

2005 ◽  
Vol 79 (2) ◽  
pp. 696-704 ◽  
Author(s):  
Anja Seybert ◽  
Clara C. Posthuma ◽  
Leonie C. van Dinten ◽  
Eric J. Snijder ◽  
Alexander E. Gorbalenya ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Enzymatic Activities ◽  
Equine Arteritis Virus ◽  
Zinc Binding ◽  
Helicase Domain ◽  
Critical Function ◽  
Binding Function ◽  
Mutant Forms

ABSTRACT Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

scholarly journals Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

2006 ◽  
Vol 80 (21) ◽  
pp. 10900-10906 ◽  
Author(s):  
Fernando Almazán ◽  
Marta L. DeDiego ◽  
Carmen Galán ◽  
David Escors ◽  
Enrique Álvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cdna Clone ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Genetic System ◽  
Infectious Cdna Clone ◽  
Reverse Genetic System ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Processing Enzymes

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

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

2000 ◽  
Vol 74 (20) ◽  
pp. 9586-9593 ◽  
Author(s):  
Anja Seybert ◽  
Leonie C. van Dinten ◽  
Eric J. Snijder ◽  
John Ziebuhr
Keyword(s):  
Functional Relationship ◽  
Biochemical Characterization ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Biochemical Properties ◽  
Equine Arteritis Virus ◽  
Helicase Domain ◽  
Rna And Dna ◽  
Combined Data

ABSTRACT 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.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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