The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Dinten, Leonie C. van; Tol, Hans van; Gorbalenya, Alexander E.; Snijder, Eric J.

doi:10.1128/jvi.74.11.5213-5223.2000

Cited by 100 publications

(136 citation statements)

References 74 publications

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“…Both coronavirus and arterivirus helicases require an N-terminal (putative) metal-binding domain, consisting of at least 12 conserved Cys and His residues, for activity. The domain is essential for replication, transcription and virion morphogenesis (as demonstrated for EAV) (van Dinten et al, 2000). The SARS-CoV helicase is predicted to be released from pp1ab by 3CL pro -mediated cleavages of the 5301 Gln|Ala 5302 and 5902 Gln|Ala 5903 dipeptide bonds ( Fig.…”

Section: Essential Proteins Involved In Replication and Transcriptionmentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

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A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and posttranslational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries. INTRODUCTIONSevere acute respiratory syndrome (SARS) is a lifethreatening form of pneumonia (Peiris et al., 2003a). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal-oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b;Drosten et al., 2003;Ksiazek et al., 2003;Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29?7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003;Marra et al., 2003;Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001;Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the 21 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et ...

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Section: Essential Proteins Involved In Replication and Transcriptionmentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

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814

960

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Section: The Minichromosome Maintenance (Mcm)mentioning

confidence: 99%

“…1) in a nonconserved region of the MCM family of proteins suggests the importance of the zinc finger domain in MCM function (2). Furthermore, mutational analyses of zinc finger domains in several viral helicases have shown these motifs to be important for helicase activity (28,29).Archaea, the third domain of life (30), are believed to replicate DNA in a eukaryotic-like fashion (31, 32). This conclusion is based in large part on the complete genome sequences of several members of this domain and the biochemical properties of the replication proteins that have been studied.…”

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confidence: 99%

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

Poplawski¹,

Grabowski²,

Long³

et al. 2001

Journal of Biological Chemistry

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The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum ⌬H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX 2 CX n CX 2 C (C 4 ) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif. The minichromosome maintenance (MCM)1 genes encode a family of six proteins (MCM2 to -7) first identified by their essential role in the maintenance of ARS-containing minichromosomes in Saccharomyces cerevisiae (1, 2). MCM homologues have been identified in all eukaryotes, and all are essential for DNA replication (1, 3-7). The recruitment of these proteins onto replication origins during the G 1 phase of the cell cycle is essential for the formation of a prereplicative complex and initiation of DNA synthesis (8 -12). In addition to the heterohexameric structure MCM2/3/4/5/6/7, found in vivo and in vitro, several additional MCM complexes have been identified (13-16). Biochemical studies in yeast and mammals have shown that a dimeric complex of the MCM4/6/7 heterotrimer contains 3Ј-5Ј DNA helicase activity, single-stranded DNA (ssDNA) binding, and DNA-dependent ATPase activities (15,17,18). Both MCM2 and MCM3/5 were shown to inhibit the helicase activity of MCM4/6/7 and thus were suggested to play regulatory roles (15,19). Both genetic and biochemical data suggest that the MCM4/6/7 complex is the eukaryotic replicative helicase and that the other MCM polypeptides may play regulatory roles (15,16,19).Zinc fingers were first identified as zinc-binding domains important for protein-DNA interactions (20, 21). They contain conserved Cys and His residues, and to date more than 10 classes have been biochemically characterized, and their structures have been determined (reviewed in Refs. 22-25). Since their discovery, zinc finger domains have been implicated in diverse functions, including ssDNA and double-stranded DNA (dsDNA) binding, RNA binding, and protein-protein interactions (reviewed in Refs. 24 and 26). The primary amino acid sequences of the eukaryotic MCM2, -4, -6, and -7 proteins from all organisms contain a putative zinc finger motif of the C 4 type, either CX 2 CX n CX 2 C (MCM2) or CX 2 CX n CX 4 C (MCM4, -6, an...

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“…For example, both the T7 and HSV helicases have zinc binding domains that help facilitate primer synthesis by their attached primases, and the HCV helicase has a zinc ion bound to its N-terminal protease region. When the zinc binding domain attached to nidovirus helicases is altered using site-directed mutagenesis, the virus displays defective RNA synthesis in infected cells [58] and the helicase is not fully active [59].…”

Section: Sars Coronavirus Helicasementioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

103

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Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

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The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Cited by 100 publications

References 74 publications

Mechanisms and enzymes involved in SARS coronavirus genome expression

Mechanisms and enzymes involved in SARS coronavirus genome expression

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

Understanding Helicases as a Means of Virus Control

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