The origins of SARS-CoV-2: A critical review.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Insights into ubiquitin chain architecture using Ub-clipping.

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies.

In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all ß-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of ß-sheets. The A46(1-83) structure itself is a ß-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

Characterization and structure of the vaccinia virus NF-κB antagonist A46.

Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-ß (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87-229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low µm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity.

The leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor.

The leader proteinase (Lpro) of the foot-and-mouth disease virus inhibits the host innate immune response by at least three different mechanisms. The most well-characterised of these is the prevention of the synthesis of cytokines such as interferons immediately after infection, brought about by specific proteolytic cleavage of the eukaryotic initiation factor 4G. This prevents the recruitment of capped cellular mRNA; however, the viral RNA can be translated under these conditions. The two other mechanisms are the induction of NF-κB cleavage and the deubiquitination of immune signalling molecules. This review focuses on the structure-function relationships in Lpro responsible for these widely divergent activities.

Strategies for purifying variants of human rhinovirus 14 2C protein.

The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.

AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities.

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

Specificity of human rhinovirus 2A(pro) is determined by combined spatial properties of four cleavage site residues.

The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

Virus species polemics: 14 senior virologists oppose a proposed change to the ICTV definition of virus species.

The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) has recently decided to modify the current definition of virus species (Code of Virus Classification and Nomenclature Rule 3.21) and will soon ask the full ICTV membership (189 voting members) to ratify the proposed controversial change. In this discussion paper, 14 senior virologists, including six Life members of the ICTV, compare the present and proposed new definition and recommend that the existing definition of virus species should be retained. Since the pros and cons of the proposal posted on the ICTV website are not widely consulted, the arguments are summarized here in order to reach a wider audience.