The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond

Ziebuhr, John; Thiel, Volker; Gorbalenya, Alexander E.

doi:10.1074/jbc.m104097200

Cited by 133 publications

(215 citation statements)

References 54 publications

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“…5). The poor conservation of HCoV-229E PL pro cleavage sites probably results from the overlapping substrate specificities of PL1 pro and PL2 pro , which we reported previously (Ziebuhr et al, 2001). Consistent with the hypothesis that conservation of only one PL pro domain is linked to an increase in specificity, we also find that the IBV PL2 pro cleavage sites are much better conserved than the corresponding HCoV-229E PL1 pro /PL2 pro sites (Fig.…”

Section: Polyprotein Processing By Pl2 Prosupporting

confidence: 78%

“…3). Except for infectious bronchitis virus (IBV) (Liu et al, 1995;Lim et al, 2000), all previously characterized coronaviruses encode two (paralogous) papain-like cysteine proteinases (PL1 pro and PL2 pro ), which cleave the N-proximal polyprotein regions at three sites (Gorbalenya et al, 1991;Bonilla et al, 1997;Baker et al, 1989;Kanjanahaluethai & Baker, 2000;Ziebuhr et al, 2001;Herold et al, 1998), while the 3C-like cysteine proteinase (3CL pro ; also called main proteinase, M pro ) cleaves the central and C-proximal regions at 11 conserved sites (Ziebuhr et al, 2000). The conservation of both the positions and sequences of coronavirus pp1a/pp1ab cleavage sites allows the substrate specificities of proteinases of newly identified coronaviruses to be predicted (Fig.…”

Section: Polyprotein Processing By Pl2 Promentioning

confidence: 99%

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Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

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815

960

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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: Polyprotein Processing By Pl2 Prosupporting

confidence: 78%

Section: Polyprotein Processing By Pl2 Promentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

Self Cite

815

960

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“…The conformation of the cysteine side chains and the arrangement of the ␤-hairpins classify this zinc-binding site as a member of the circularly permutated, zinc-ribbon fold group (22). Biochemical evidence, computer modeling, and bioinformatics have predicted the presence of zinc-binding domains in coronavirus PLpros (19,23), and we have demonstrated, through mutational analysis of the zinc-coordinating cysteines of SARSCoV PLpro, that zinc-binding ability is essential for structural integrity and protease activity (17).…”

Section: Resultsmentioning

confidence: 99%

“…Both tyrosines are conserved in HAUSP and USP14. Coronavirus PLP2-like enzymes with substrate specificities requiring a glycine at P1 and either a glycine or alanine at P2 also contain a tyrosine at a position analogous to Y274 and a bulky aromatic group at a position corresponding to Y113 (23,31,32). It is also important to note that aromatic residues neighboring the catalytic cysteine are very common in PLpros because they can serve to enhance the nucleophilicity of the cysteine residue (33).…”

Section: Resultsmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Ratia

Saikatendu²,

Santarsiero

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

385

531

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Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitinlike N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitinaldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.membrane-associated protease ͉ ubiquitin-like domain

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“…Expression of pp1ab involves ribosomal frameshifting into the Ϫ1 frame just upstream of the ORF 1a translation termination codon (9). Human coronavirus polyproteins are autoproteolytically processed by two (SARSCoV) or three (HCoV-229E) viral proteases called papain-like proteases and 3C-like main protease (5,(10)(11)(12). In total, at least 16 processing end-products called nonstructural proteins (nsp) 1-16 are derived from the pp1a and pp1ab polyproteins (8).…”

mentioning

confidence: 99%

Major genetic marker of nidoviruses encodes a replicative endoribonuclease

Иванов

Hertzig²,

Rozanov³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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271

407

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Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the Ϸ30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn 2؉ -dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that doublestranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2-3 cyclic phosphate ends. 2-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription. H uman coronavirus 229E (HCoV-229E), a group 1 coronavirus, is one of the major viral pathogens causing upper respiratory tract illness in humans (1), whereas severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2 coronavirus, has been identified as the causative agent of SARS, a life-threatening form of pneumonia that caused Ͼ8,000 fatalities in a worldwide epidemic in 2003 (2, 3). The extremely large positive-strand RNA genomes of HCoV-229E (27.3 kb) and SARS-CoV (29.7 kb) contain 8 and 14 ORFs, respectively (4, 5). The two most 5Ј-terminal ORFs, 1a and 1b, encode the major subunits of the replicative machinery, whereas the more downstream ORFs encode structural and virus-specific accessory proteins.Coronavirus gene expression involves a series of complex transcriptional, translational, and posttranslational regulatory mechanisms (6, 7). After receptor-mediated entry, two large replicative polyproteins, pp1a (Ͼ450 kDa) and pp1ab (Ͼ750 kDa), are translated from the genome RNA. The polyproteins are encoded by the replicase gene (Ͼ20,000 bases) that comprise ORFs 1a and 1b (Fig. 1A) (8). Expression of pp1ab involves ribosomal frameshifting into...

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The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond

Cited by 133 publications

References 54 publications

Mechanisms and enzymes involved in SARS coronavirus genome expression

Mechanisms and enzymes involved in SARS coronavirus genome expression

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Major genetic marker of nidoviruses encodes a replicative endoribonuclease

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