John Ziebuhr

Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.

Abstract The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

Virus-encoded proteinases and proteolytic processing in the Nidovirales

IP: 54.191.40.80 On: Sun, 20 Aug 2017 08:42:34 Journal of General Virology (2000), 81, 853–879. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nidovirales: Evolving the largest RNA virus genome

Abstract This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3′-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV); Announcement of the Coronavirus Study Group

During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus (CoV) was isolated from the sputum of a patient with acute pneumonia and renal failure ([1][1], [2][2]). The isolate was provisionally called human coronavirus Erasmus Medical Center (EMC) ([3][3]). Shortly thereafter

Ribose 2′- O -methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5′ cap structures of higher eukaryote mRNAs have ribose 2′-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2′-O-methylation of mRNA remains elusive. Here we show that 2′-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2′-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2′-O-methylation of mRNA suggests that RNA modifications such as 2′-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, Mpro, a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus Mpro is reported. The structure was refined to 1.96 Å resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that Mpro self‐processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin‐like fold that is connected by a 16 residue loop to an extra domain featuring a novel α‐helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of Mpro substrates. Interactions involving the N‐terminus and the α‐helical domain stabilize the loop in the orientation required for trans‐cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

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.

The coronavirus replicase.

Coronavirus genome replication and transcription take place at cytoplasmic membranes and involve coordinated processes of both continuous and discontinuous RNA synthesis that are mediated by the viral replicase, a huge protein complex encoded by the 20-kb replicase gene. The replicase complex is believed to be comprised of up to 16 viral subunits and a number of cellular proteins. Besides RNA-dependent RNA polymerase, RNA helicase, and protease activities, which are common to RNA viruses, the coronavirus replicase was recently predicted to employ a variety of RNA processing enzymes that are not (or extremely rarely) found in other RNA viruses and include putative sequence-specific endoribonuclease, 3′-to-5′ exoribonuclease, 2′-O-ribose methyltransferase, ADP ribose 1′-phosphatase and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase activities. This chapter reviews (1) the organization of the coronavirus replicase gene, (2) the proteolytic processing of the replicase by viral proteases, (3) the available functional and structural information on individual subunits of the replicase, such as proteases, RNA helicase, and the RNA-dependent RNA polymerase, and (4) the subcellular localization of coronavirus proteins involved in RNA synthesis. Although many molecular details of the coronavirus life cycle remain to be investigated, the available information suggests that these viruses and their distant nidovirus relatives employ a unique collection of enzymatic activities and other protein functions to synthesize a set of 5′-leader-containing subgenomic mRNAs and to replicate the largest RNA virus genomes currently known.

Molecular biology of severe acute respiratory syndrome coronavirus

The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. Coronaviruses and their closest relatives possess extremely large plus-strand RNA genomes and employ unique mechanisms and enzymes in RNA synthesis that separate them from all other RNA viruses. The SARS epidemic prompted a variety of studies on multiple aspects of the coronavirus replication cycle, yielding both rapid identification of the entry mechanisms of SARS-CoV into host cells and valuable structural and functional information on SARS-CoV proteins. These recent advances in coronavirus research have important implications for the development of anti-SARS drugs and vaccines.

Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology.

Summary Many viruses induce hepatitis in humans, highlighting the need to understand the underlying mechanisms of virus-induced liver pathology. The murine coronavirus, mouse hepatitis virus (MHV), causes acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV accessory protein, ns2, antagonizes the type I interferon response and promotes hepatitis. We show that ns2 has 2′,5′-phosphodiesterase activity, which blocks the interferon inducible 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway to facilitate hepatitis development. Ns2 cleaves 2′,5′-oligoadenylate, the product of OAS, to prevent activation of the cellular endoribonuclease RNase L and consequently block viral RNA degradation. An ns2 mutant virus was unable to replicate in the liver or induce hepatitis in wild-type mice, but was highly pathogenic in RNase L deficient mice. Thus, RNase L is a critical cellular factor for protection against viral infection of the liver and the resulting hepatitis.