Alexander E. Gorbalenya

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.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015)

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

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

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae.

Summary. The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22–25% and 21–25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63–71% and 59–67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

Severe acute respiratory syndrome coronavirus phylogeny: toward consensus.

Since the identification of a new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) as the causative agent of the SARS epidemic in the winter of 2002-2003, the origin of the novel agent has remained a hotly debated topic. Which virus was the immediate ancestor of SARS-CoV, and what are the relationships between SARS-CoV and other previously described coronaviruses? Correct answers to these two questions are vital, as substantiated below, for designing strategies to detect, contain, and combat new outbreaks and for dissecting the fundamentals of the SARS-CoV life cycle. Major efforts have been invested in a thus far unsuccessful search for a natural SARS-CoV reservoir. In the meantime, and more outside the spotlight, SARS-CoV genome sequences have been used to define the phylogenetic position of SARSCoV among coronaviruses. These studies have resulted in a lot of controversy whose intricacies may not be very clear to outsiders. Our purpose is to clarify the situation from an insider’s point of view. Originally, coronaviruses were classified on the basis of antigenic cross-reactivity, and in this manner three antigenic groups (1 to 3) were recognized (14). When coronavirus genome sequences began to accumulate, the same groups were evident from phylogenetic analyses of the four structural proteins, N, M, E, and S (19), and of different regions of the giant replicase (3, 22). Group boundaries were also supported by the diversity of small open reading frames (ORFs) encoding accessory proteins, which are dispersed among the structural protein genes in the 3 -proximal region of the genome (Fig. 1). In the middle of the nineties, a first discord between the antigenicity-based and phylogenetic classifications emerged upon the characterization of the coronavirus porcine epidemic diarrhea virus (PEDV) and human coronavirus 229E (HCoV229E), one of the common cold viruses. These viruses proved not to have antigenic cross-reactivity with members of the established groups (18), yet on the basis of sequence comparisons it was concluded that they segregate into group 1, although they are somewhat separated from porcine transmissible gastroenteritis virus and closely related viruses (subgroup 1b and subgroup 1a, respectively, in Fig. 2) (9). The PEDV and HCoV-229E genomes also share an ORF specific for group 1 in the 3 -proximal region of their genome. The Coronavirus Study Group of the International Committee on Taxonomy of Viruses recognized these viruses as members of group 1 rather than declaring them prototypes of new groups (6). This decision effectively converted the original antigenic groups—which were based essentially on some properties of one or a few viral proteins—into a genetic one based on full-length genome sequences, but this change was never acknowledged explicitly. Consequently, no guidelines were established with respect to handling future disagreements between the classifications based on antigenicity, genome organization, and phylogeny should these arise from the properties of newly identified coronaviruses, and SARS-CoV proved to be quite a classification challenge. Initial phylogenetic analyses suggested that the novel virus did not cluster with any of the three established coronavirus groups. Accordingly, SARS-CoV also has a unique pattern of small ORFs in the 3 -proximal region of its genome and a unique internal organization of its nonstructural protein 3 (nsp3) replicase subunit, which includes a sizable novel domain (SARS-CoV unique domain SUD) and only one papain-like protease (PL2pro) rather than the two copies commonly found in other coronaviruses (Fig. 1). Although a thorough assessment of the antigenic cross-reactivity of SARS-CoV with other coronaviruses is yet to be published, a proposal to recognize SARS-CoV as a representative of a new, fourth group of coronaviruses seemed most logical (15, 17). If SARS-CoV indeed represents a new group, then when, relative to other groups, could this lineage have emerged? Several scenarios are theoretically plausible, and one of the most extreme ones, which seems compatible with the unique characteristics of SARS-CoV, places the origin of this lineage next to the ancestor of the other coronaviruses (Fig. 2A). To rigorously infer the origin of SARS-CoV, we conducted a special analysis of the replicase ORF1b region (Fig. 1), the mostconserved part of the coronavirus genome, which accounts for 20% of its size (20). In this analysis, the equine torovirus—a distant relative of coronaviruses belonging to the genus Torovirus of the same Coronaviridae family—was used as an outgroup to infer the direction of coronavirus evolution. Surprisingly, our fully resolved tree demonstrated that the SARS-CoV lineage is an early split-off from the group 2 branch and that the split-off occurred relatively late in coronavirus evolution, after the two bifurcations that gave rise to the three previously established groups (Fig. 2B). This topology is unlikely to be skewed, as it was obtained by using different criteria * Corresponding author. Mailing address: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, E4-P, Room L04-036, 2333 ZA Leiden, The Netherlands. Phone: 31-71-526-1652. Fax: 31-71-526-6761. E-mail: a.e.gorbalenya@lumc.nl.

SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus

In (+) RNA coronaviruses, replication and transcription of the giant ∼30 kb genome to produce genome‐ and subgenome‐size RNAs of both polarities are mediated by a cognate membrane‐bound enzymatic complex. Its RNA‐dependent RNA polymerase (RdRp) activity appears to be supplied by non‐structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5′‐(G/U)CC‐3′ trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C‐terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA‐dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer‐dependent nsp12 RdRp.

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.

Coronavirus Nonstructural Protein 16 Is a Cap-0 Binding Enzyme Possessing (Nucleoside-2′O)-Methyltransferase Activity

ABSTRACT The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-l-methionine (AdoMet)-dependent RNA (nucleoside-2′O)-methyltransferase (2′O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap (7MeGpppAC3-6) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2′O position of the first transcribed nucleotide, thus converting 7MeGpppAC3-6 into 7MeGpppA2′OMeC3-6. The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2′O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2′O methylation follows an order of events in which 2′O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

Molecular analysis of three Ljungan virus isolates reveals a new, close-to-root lineage of the Picornaviridae with a cluster of two unrelated 2A proteins.

ABSTRACT Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles (Clethrionomys glareolus). In the present study, it is revealed through comparative sequence analysis that three newly determined Swedish LV genomes are closely related and possess a deviant picornavirus-like organization: 5′ untranslated region-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3′ untranslated region. The LV genomes and the polyproteins encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho-, and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses, and avian encephalomyelitis virus. The unprecedented association of two structurally different 2A proteins is a feature never previously observed among picornaviruses and implies that their functions are not mutually exclusive. Secondary polyprotein processing of the LV polyprotein is mediated by proteinase 3C (3Cpro) possessing canonical affinity to Glu and Gln at the P1 position and small amino acid residues at the P1′ position. In addition, LV 3Cpro appears to have unique substrate specificity to Asn, Gln, and Asp and to bulky hydrophobic residues at the P2 and P4 positions, respectively. Phylogenetic analysis suggests that LVs form a separate division, which, together with the Parechovirus genus, has branched off the picornavirus tree most closely to its root. The presence of two 2A proteins indicates that some contemporary picornaviruses with a single 2A may have evolved from the ancestral multi-2A picornavirus.