Epistemic_Statements  

CORD-19:ba334a8f734285257878e0df4e64a6131818d0c6 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-75 Epistemic_statement denotes Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes
T2 86-204 Epistemic_statement denotes Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales.
T3 1009-1180 Epistemic_statement denotes In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase).
T4 1181-1385 Epistemic_statement denotes Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade.
T5 1899-2201 Epistemic_statement denotes Positive-stranded RNA (+RNA) viruses that belong to the order Nidovirales infect a wide range of vertebrates (families Arteriviridae and Coronaviridae) or invertebrates (Mesoniviridae and Roniviridae) (de Groot et al., 2012; Lauber et al., 2012) and can have a significant economic and societal impact.
T6 2202-2706 Epistemic_statement denotes For example, infections with the arterivirus (AV) porcine reproductive and respiratory syndrome virus (PRRSV) have severely affected the swine industry for almost three decades now (Holtkamp et al., 2013; Perez et al., 2015; Pileri and Mateu, 2016) , whereas zoonotic coronaviruses (CoVs) have caused episodes of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in humans (Graham et al., 2013; Perlman and Netland, 2009 ) and may do so again (Menachery et al., 2015) .
T7 3235-3435 Epistemic_statement denotes However, nidovirus genome sizes vary significantly, with AV genomes ranging from 13 to 16 kilobases (kb), mesonivirus genomes from 20 to 21 kb, and CoV genomes from 26 to 34 kb (Lauber et al., 2013) .
T8 5058-5195 Epistemic_statement denotes Nidovirus genomes are thought to have expanded gradually by gene duplication and the acquisition of novel domains (Lauber et al., 2013) .
T9 5196-5410 Epistemic_statement denotes During this process, specific innovations may have enabled them to explore an unprecedented evolutionary space and adapt to a wide variety of host organisms, including mammals, birds, fish, insects and crustaceans.
T10 5411-5762 Epistemic_statement denotes In particular, the general genome size restrictions of RNA viruses, commonly attributed to the poor nucleotide incorporation fidelity of the viral RdRp domain, may have been mitigated by the acquisition of compensatory enzymatic functions (Deng et al., 2014; Eckerle et al., 2010; Eckerle et al., 2007; Gorbalenya et al., 2006; Snijder et al., 2003) .
T11 6313-6447 Epistemic_statement denotes The RNA-templated synthesis of new RNA by the viral RNA polymerase is arguably the key step in the infection cycle of all RNA viruses.
T12 7045-7220 Epistemic_statement denotes For the purpose of this review, we will refer to the process of sg mRNA synthesis as 'transcription'; the underlying mechanisms will be discussed in more detail in Section 10.
T13 7304-7571 Epistemic_statement denotes Minus strand RNA synthesis can be either continuous (producing a full-length minus-strand template for genome replication) or discontinuous to produce the subgenome-length templates for the production of the sg mRNAs (Sawicki and Sawicki, 1995; Sethna et al., 1989) .
T14 7572-7901 Epistemic_statement denotes In addition to the overall RNA structure of the genome and transcription-specific protein factors, conserved transcription-regulatory sequences (TRSs) in the genomic template are thought to be the prime trigger and guiding elements that make the nidovirus RTC synthesize a subgenome-length rather than a full-length minus strand.
T15 7902-8223 Epistemic_statement denotes Thus, the TRSs constitute one class of cis-acting RNA signals with which the nidovirus RTC needs to interact, although we note that this feature has mainly been addressed from the RNA side (e.g., by sitedirected mutagenesis of TRSs) (Dufour et al., 2011; Pasternak et al., 2001; Zheng et al., 2014; Zuniga et al., 2004) .
T16 8653-8831 Epistemic_statement denotes For each genome, the known open reading frames (ORFs) are indicated with the replicase ORFs 1a and 1b depicted in grey and structural protein genes depicted in different colours.
T17 9978-10094 Epistemic_statement denotes The zinc-finger domain (F) in EAV nsp1 that is crucial for subgenomic mRNA synthesis (see text) is also highlighted.
T18 10949-11146 Epistemic_statement denotes However, it has remained largely unclear whether these elements interact with the RdRp domain-containing subunit directly or with other components of the RTC, as for example reported by Züst et al.
T19 11147-11385 Epistemic_statement denotes who observed that second-site mutations in the small nsp8 and nsp9 subunits of the RTC could compensate for mutations in conserved RNA sequences near the genomic 3 end of the murine hepatitis coronavirus (MHV) genome (Züst et al., 2008) .
T20 11386-11701 Epistemic_statement denotes In the case of the 5 -proximal domain of the genomic RNA (or its minus-stranded counterpart), functional studies have been complicated by the fact that RNA signals for replication, transcription, translation and (potentially) packaging may be overlapping and difficult to separate, both physically and functionally.
T21 11702-11874 Epistemic_statement denotes In this review, we will focus mainly on 'the protein side' of nidovirus RNA synthesis, and what is known about the initiation of RNA synthesis by nidovirus RNA polymerases.
T22 12085-12286 Epistemic_statement denotes These replicase subunits, which also carry a unique and nidovirus-specific N-terminal extension, are believed to catalyse the nucleotide condensation reaction that drives replication and transcription.
T23 13084-13337 Epistemic_statement denotes Although the presence of this N-terminal extension was first recognized when the replicase polyprotein cleavage sites were predicted over 25 years ago (Gorbalenya et al., 1989) , an enzymatic activity (nucleotidylation) was assigned to it only recently.
T24 13529-13673 Epistemic_statement denotes As the Cterminal border of NiRAN is difficult to define, yet another domain (approximately 50-175 amino acids for AV and CoV, respectively; Fig.
T25 13674-13755 Epistemic_statement denotes 2A ) may turn out to connect the NiRAN and RdRp domains (Lehmann et al., 2015a) .
T26 15412-15561 Epistemic_statement denotes 3B ), yet experimental studies to support this model have been greatly hampered by difficulties with attempts to stably express and purify CoV nsp12.
T27 16855-17027 Epistemic_statement denotes However, using sequence alignments, secondary structure predictions and homology modelling, the molecular structure of the nsp12 subunit of SARS-CoV (Xu et al., 2003) (Fig.
T28 17028-17082 Epistemic_statement denotes 3B ) and MHV (Sexton et al., 2016) has been predicted.
T29 17083-17355 Epistemic_statement denotes In case of the former, a model could be reliably generated for its conserved RdRp domain, based on a careful alignment of amino acids 388-890 to known polymerase sequences (Xu et al., 2003) An analysis of the predicted CoV RdRp domain architecture using three models (Fig.
T30 17742-17981 Epistemic_statement denotes The duplex that is formed as the RNA polymerase catalyses nucleotide condensation, thus consisting of the nascent RNA product and the viral template, likely leaves the enzyme via a relatively wide exit channel at the front of the molecule.
T31 17982-18344 Epistemic_statement denotes This predicted three-channel architecture would make the nidovirus RNA polymerase comparable to other +RNA-viral RNA polymerases, but different from the RNA polymerases of negative-stranded RNA viruses, which are believed to have separate exit channels for the template and nascent strand (Pflug et al., 2017; Reguera et al., 2016; te Velthuis and Fodor, 2016) .
T32 18345-18549 Epistemic_statement denotes Putative channels similar to those predicted for the CoV nsp12 RdRp domain can be seen in the models of the EAV nsp9 RdRp domain generated with the prediction tools Swiss-Model, Phyre2 and I-TASSER ( Fig.
T33 18550-18712 Epistemic_statement denotes 3C ), in line with earlier sequence analyses suggesting that the overall molecular architecture is conserved among nidovirus RdRp domains (Beerens et al., 2007) .
T34 18953-19135 Epistemic_statement denotes This is in line with the presence of (a partial) motif G (see above) and together these features may have important functional implications for the initiation of viral RNA synthesis.
T35 19447-19634 Epistemic_statement denotes As described for other RdRp domains, the key lysine of motif D, which likely acts as general acid during catalysis (Castro et al., 2009) , is located near the entrance of the NTP channel.
T36 20107-20329 Epistemic_statement denotes Subsequent mutation of these residues to isoleucine (V553I) and phenylalanine (M611F) in the virus conferred resistance to 5-fluorouracil (5-FU) and 5-azacytidine (5-AZC), or 5-FU only, respectively (Sexton et al., 2016) .
T37 20891-21227 Epistemic_statement denotes Bacterial expression of the latter (i.e., ub-nsp12-His 6 ) still resulted in an unstable protein, but co-expression of the ubiquitin protease Ubp1, which can hydrolyse the ub-nsp12 fusion site to produce a recombinant nsp12-His 6 containing the natural nsp12 N-terminus, significantly improved its stability (te Velthuis et al., 2010a).
T38 21360-21688 Epistemic_statement denotes Overall, these results suggested that, as for the poliovirus RNA polymerase 3D pol (Thompson and Peersen, 2004) , the N-terminus of nsp12 or, alternatively, the proper folding of the NiRAN domain which is not present in 3D pol , is important for the stability and possibly also the activity of nsp12 (te Velthuis et al., 2010a).
T39 21689-21919 Epistemic_statement denotes Analysis of the CoV nsp12 amino acid sequence using alignments and molecular modelling predicted that the enzyme lacks a priming loop or other initiation platform that would promote de novo initiation of RNA synthesis (see above).
T40 22071-22175 Epistemic_statement denotes 2A and 3B), it was assumed that a primer would be required for the initiation of RNA synthesis by nsp12.
T41 23648-23750 Epistemic_statement denotes regarding the constructs and templates used) may have contributed to the mostly contradictory results.
T42 23751-23962 Epistemic_statement denotes Nevertheless, together the observations teach us that the activity of nsp12 alone must be relatively weak and sensitive to the presence of purification tags, buffer conditions and the simplicity of the template.
T43 23963-24159 Epistemic_statement denotes It is likely that CoV nsp12 requires additional factors that improve its RNA polymerase activity and there is indeed evidence that other nsps fulfil this role (see below) (Subissi et al., 2014b) .
T44 24160-24369 Epistemic_statement denotes In a study aimed at finding putative interaction partners for nsp12, a second RNA polymerase activity was reported to be associated with SARS-CoV nsp8, a 22-kDa protein encoded in ORF1a (Imbert et al., 2006) .
T45 24370-24633 Epistemic_statement denotes This activity, which would be unique among RNA viruses, was observed to initiate RNA synthesis in a primer-independent manner on templates containing cytidine-rich sequences in vitro and to generate products of approximately 6 nt in length (Imbert et al., 2006) .
T46 24634-24860 Epistemic_statement denotes Together with the assumption based on comparative genomics that nsp12 is a primer-dependent RNA polymerase (see above), nsp8 was thus proposed to function as a primase, presumably generating the initiation substrate for nsp12.
T47 25216-25323 Epistemic_statement denotes de novo synthesis but also primer extension activity) can be activated by the removal of the terminal tags.
T48 25324-25708 Epistemic_statement denotes These observations are in line with experiments showing that nsp8 appears to be largely inactive in the context of the precursor polyproteins nsp7-8 and nsp7-10 (te Velthuis et al., 2012; Xiao et al., 2012) and together they suggest that the (faithful) processing of nsp8 can affect and potentially regulate the activity of nsp8 in the complexes in which it resides in infected cells.
T49 25709-25902 Epistemic_statement denotes We note, however, that the ability of nsp8 to extend primer-template duplexes with [␣-32 P]ATP could not be confirmed in a subsequent study using a radiolabeled primer (Subissi et al., 2014b) .
T50 25979-26189 Epistemic_statement denotes The structure of nsp8 alone is presently unknown, but it is likely that it forms oligomers based on gel shift, electron microscopy (EM) and gel filtration studies (te Velthuis et al., 2012; Zhai et al., 2005) .
T51 26190-26356 Epistemic_statement denotes The involvement of conserved nsp8 residues in its polymerase activity has so far been probed in two in vitro studies (Imbert et al., 2006; te Velthuis et al., 2012) .
T52 26818-27055 Epistemic_statement denotes The critical role of the above residues was substantiated, in part, by observations that nsp8 mutations D52A and K82A cripple SARS-CoV RNA synthesis in cell culture and that mutation K58A is lethal for the virus (Subissi et al., 2014b) .
T53 27056-27414 Epistemic_statement denotes However, nsp8 shares no structural homology with other primases or RNA polymerases (see Section 6), so in the absence of a tertiary structure of nsp8 bound to RNA and nucleotides, it is presently unclear how these putative active site residues may assist the nucleotide condensation activity of nsp8 in vitro (Imbert et al., 2006; te Velthuis et al., 2012) .
T54 27415-27702 Epistemic_statement denotes Moreover, as for SARS-CoV nsp12, contradictory results have been obtained in the handful of nsp8 studies performed by different laboratories, which may have been due to differences in experimental design and/or the fact that nidovirus RdRps appear to be technically challenging proteins.
T55 27703-27824 Epistemic_statement denotes Unfortunately, we therefore have to conclude that the question whether nsp8 functions as primase still remains wide open.
T56 27825-27942 Epistemic_statement denotes In the next section, we will elaborate on the role of nsp8 as a cofactor for RNA replication by nsp12 in more detail.
T57 28187-28362 Epistemic_statement denotes This complex consisted of 8 copies of each subunit, oriented such that the inner cavity of the ring was positively charged and capable of binding RNA (Zhai et al., 2005) (Fig.
T58 28369-28637 Epistemic_statement denotes Given the structural similarity of this hexadecamer to the eukaryotic PCNA sliding clamp and the ␤-subunit of the E. coli DNA polymerase III, it was proposed that it could function as a processivity factor for the RNA polymerase function of nsp12 (Zhai et al., 2005) .
T59 28638-28864 Epistemic_statement denotes Interestingly, nsp7 and nsp8 from the distantly related feline coronavirus (FCoV) were found to form a different nsp7+8 complex in which nsp7 and nsp8 form a heterotrimer consisting of 2 copies of nsp7 and 1 copy of nsp8 (Fig.
T60 28892-29141 Epistemic_statement denotes Although the heterotrimer was found to be the most likely complex in solution, it may be possible that this trimer forms higher order structures, as it can self-interact under crystallization conditions and form a heterohexamer (Xiao et al., 2012) .
T61 29142-29294 Epistemic_statement denotes Together with the observations from Zhai et al., this suggests that the nsp7+8 complex is relatively plastic and that it may perform multiple functions.
T62 29457-29709 Epistemic_statement denotes Alternatively, the difference between the various nsp7+8 complex structures can be attributed to the position of the purification tags, the genetic background of the protein (FCoV versus SARS-CoV) or the crystallization procedures (Xiao et al., 2012) .
T63 29710-29847 Epistemic_statement denotes In contrast to their oligomeric form, the structure of the individual nsp7 and nsp8 subunits is consistent across the structural studies.
T64 30007-30120 Epistemic_statement denotes Nsp8 can adopt a golf club-like shape that is present in a 'straight' (nsp8-I) and 'bent' (nsp8-II) conformation.
T65 30303-30469 Epistemic_statement denotes Inside the ring structure, SARS-CoV nsp7 and nsp8 interact extensively, with nsp7 likely stabilising the framework of interlinking nsp8 subunits (Zhai et al., 2005) .
T66 30470-30874 Epistemic_statement denotes Although SARS-CoV nsp8 can assemble into oligomers in the absence of nsp7 (te Velthuis et al., 2012; Zhai et al., 2005) , (Johnson et al., 2010; Xiao et al., 2012; Zhai et al., 2005) , nsp7+8 complexes were found to have a higher RNA binding affinity than nsp8 oligomers alone (te Velthuis et al., 2012) and mutations in nsp7 affect the RNA binding ability of the nsp7+8 complex (Subissi et al., 2014b) .
T67 31041-31252 Epistemic_statement denotes discovered that an unknown form of the nsp7+8 complex can interact with nsp12-strep and increase the processivity of its primer-dependent polymerase activity several orders of magnitude (Subissi et al., 2014b) .
T68 31467-31763 Epistemic_statement denotes Interestingly, the efficiency of the polymerase reaction was improved by creating a physical link between the nsp7 and nsp8 subunits in the form of a 6-or 12-amino acid linker (nsp7L8), which suggests that nsp7+8 complex formation on the template is a rate-limiting step (Subissi et al., 2014b) .
T69 31975-32129 Epistemic_statement denotes Unfortunately, no direct comparison was made between nsp7+8 and nsp7L8, so it is unclear whether the nsp7+8+12-strep complex behaves in a similar fashion.
T70 32529-32820 Epistemic_statement denotes In line with their observed roles in the complex, each of these three mutations also inactivated the primer-dependent and de novo activity of the nsp7+8+12 complex in vitro and resulted in a non-viable virus phenotype when reverseengineered into the SARS-CoV genome (Subissi et al., 2014b) .
T71 32821-33104 Epistemic_statement denotes By contrast to the above three mutations, only limited correlation between the biochemical and the reverse genetics data was observed for other mutations in nsp8, suggesting that the role of nsp7 and nsp8 in the RTC is more complex than the in vitro experiments will have us believe.
T72 33384-33464 Epistemic_statement denotes This suggests that the RdRp activity of nsp7+8 as observed by te Velthuis et al.
T73 33465-33624 Epistemic_statement denotes is not involved in the primer-extension activity of the nsp7+8+12 complex, which provides support for the hypothesis that nsp7+8 acts as a processivity factor.
T74 33625-33808 Epistemic_statement denotes However, the ability of nsp8 mutants D52A and D82A to support de novo initiation on the 3 terminus of the viral genome in the context of the nsp7+8+12 complex has not yet been tested.
T75 33876-34039 Epistemic_statement denotes 2C ) resulted in an inactive complex that showed no primerextension and, rather surprisingly, no de novo initiation activity on the 3 terminus of the viral genome.
T76 34040-34145 Epistemic_statement denotes In light of the previous biochemical studies on nsp8 and nsp12, it is difficult to interpret this result.
T77 34146-34281 Epistemic_statement denotes On the one hand, it suggests that only nsp12 can actively catalyse RNA synthesis, while nsp7 and nsp8 are merely present as co-factors.
T78 34282-34527 Epistemic_statement denotes On the other hand, it is presently just as likely that nsp12 is a dominant factor in the nsp7+8+12 complex, which regulates the primase and elongation activities of nsp8, or that nsp7+8+12 is still an incomplete or incorrectly assembled complex.
T79 34528-34759 Epistemic_statement denotes In turn, the lack of one or more additional (protein or RNA) factors may prevent us from seeing consistency between the biochemical responses of the polymerase complex in vitro and the functionality of the RTC in the infected cell.
T80 34829-34898 Epistemic_statement denotes and the initiation mechanism employed by nsp12 require further study.
T81 34899-35060 Epistemic_statement denotes A) The hollow hexadecameric ring of the SARS-CoV nsp7-8 complex has a positively charged channel (blue surface shading) that is likely important for RNA binding.
T82 35310-35406 Epistemic_statement denotes The nsp8 structure can adopt two conformations, here shaded green (nsp8-I) and orange (nsp8-II).
T83 35800-35905 Epistemic_statement denotes To our knowledge, attempts to achieve in vitro activity of an AV RdRp have only been made using EAV nsp9.
T84 35906-36102 Epistemic_statement denotes By contrast to SARS-CoV nsp12, recombinant EAV nsp9 with a C-terminal Histag could be stably expressed to high levels in E. coli and readily purified (Beerens et al., 2007; Lehmann et al., 2016) .
T85 36253-36444 Epistemic_statement denotes However, no activity was found when an RNA template representing the 3 -terminal part of the viral genome was provided or when a primer was hybridized to the template (Beerens et al., 2007) .
T86 36445-36581 Epistemic_statement denotes On the homopolymeric templates, RNA synthesis required the presence of Mn 2+ , but it could be stimulated by Mg 2+ if Mn 2+ was present.
T87 37100-37381 Epistemic_statement denotes Sufficient C-terminally His 6 -tagged nsp9 could be purified, but after extensive mutation of the conserved motif A and C aspartates the protein preparations continued to show de novo and primer-dependent polymerase activities on both RNA and DNA templates (Lehmann et al., 2016) .
T88 37382-37490 Epistemic_statement denotes It was thus concluded that contaminating trace amounts of T7 RNA polymerase may have produced these results.
T89 37491-37698 Epistemic_statement denotes To alleviate this problem, nsp9 was expressed in a T7 polymerase-free E. coli system that had previously been used to express a C-terminally tagged nsp12 with a native N-terminus (te Velthuis et al., 2010a).
T90 37948-38200 Epistemic_statement denotes It must therefore be concluded that, as for SARS-CoV nsp12, the activity of nsp9 likely is very weak and sensitive to the purification or assay conditions, and that -by analogy with CoV nsp12 -other co-factors may be required to stimulate its activity.
T91 38201-38424 Epistemic_statement denotes However, preliminary experiments in which RdRp assays with recombinant EAV nsp9 were supplemented with several small products from the nsp6-8 region of pp1a failed to activate RNA synthesis in vitro (Lehmann et al., 2016) .
T92 38425-38620 Epistemic_statement denotes It has long been recognized that the nidovirus replicase subunit that harbours the RdRp domain has an unusually large N-terminal extension that does not seem to be part of the RdRp domain itself.
T93 38621-38885 Epistemic_statement denotes No viral or cellular homologues have been identified thus far, and even within the order Nidovirales the level of conservation of this domain is very limited, which long hampered attempts to deduce a possible function and/or activity by using bioinformatics tools.
T94 39100-39383 Epistemic_statement denotes The domain is only present in nidoviruses and, in addition to a unique zinc-binding domain (ZBD) that is associated with the nidovirus helicase (Deng et al., 2014) , is considered to be a second universal genetic marker for this virus order, and the first with an enzymatic activity.
T95 39384-39666 Epistemic_statement denotes Despite limited sequence conservation and significant size differences, from ∼220 residues in AV to over 300 residues in CoV, three motifs were identified: A N , B N and C N (the subscript N for the NiRAN domain was added to discriminate them from the conserved RdRp domain motifs).
T96 39999-40245 Epistemic_statement denotes The C-terminal border of the domain could not be defined with certainty leaving room for the presence of a third domain of ∼50 or ∼175 amino acids for AV and CoV, respectively, that may connect the NiRAN and RdRp domains (Lehmann et al., 2015a) .
T97 40721-40840 Epistemic_statement denotes Most likely a phosphoamide bond is formed between the nucleotide and a conserved lysine in motif A N (K54 in EAV nsp9).
T98 40841-41076 Epistemic_statement denotes Mutagenesis of conserved residues in motifs A N , B N or C N resulted in less than 10% in vitro activity for all but the K106A mutant, which confirmed the association of the nucleotidylation activity with the N-terminal domain of nsp9.
T99 41077-41252 Epistemic_statement denotes A D445A mutation in motif A of the nsp9 RdRp domain only moderately affected nucleotidylation, strengthening the notion that this activity is associated with the NiRAN domain.
T100 41563-41730 Epistemic_statement denotes Despite convincing evidence for the nucleotidylation activity of EAV nsp9, this activity is yet to be demonstrated for CoVs and other members of the order Nidovirales.
T101 41731-41821 Epistemic_statement denotes In addition, the role of this activity in the nidovirus replicative cycle remains unknown.
T102 41822-42050 Epistemic_statement denotes Given its covalent linkage to the RdRp domain, it has been suggested that NiRAN is important for RNA synthesis, in analogy to the N-terminal domain of the RdRp of double-stranded RNA viruses (Tao et al., 2002; Xu et al., 2003) .
T103 42051-42248 Epistemic_statement denotes It may also add to the stability of the NiRAN and RdRp-containing replicase subunit, as suggested by the inability to express EAV nsp9 when the N-terminal domain is deleted (Beerens et al., 2007) .
T104 42249-42485 Epistemic_statement denotes While an RdRp mutation moderately influenced NiRAN nucleotidylation, it was unfortunately not possible to study the effect of NiRAN mutations on AV RNA polymerase activity, as a robust in vitro assay remains to be developed (see above).
T105 42486-42661 Epistemic_statement denotes Yet, given the integration of the two domains in a single replicase subunit, structural and/or functional crosstalk between the NiRAN and the RdRp domain is a likely scenario.
T106 42662-42818 Epistemic_statement denotes So far, three possible functions have been proposed for the nucleotidylation activity of the NiRAN domain in nidoviral replication (Lehmann et al., 2015a) .
T107 42819-43047 Epistemic_statement denotes The first is a role in the ligation of yet to be identified RNA molecules based on the domain's ability to covalently bind nucleotide monophosphates, one of the steps of the universal ligation mechanism (Shuman and Lima, 2004) .
T108 43048-43297 Epistemic_statement denotes A second is a possible role in the 5 capping of mRNAs, while a third possibility is that the NiRAN domain is involved in facilitating protein-primed RNA synthesis, similar to the use of a uridylated VpG primer by picornaviruses (Paul et al., 2000) .
T109 43298-43374 Epistemic_statement denotes Each of these hypotheses has been discussed in more detail by Lehmann et al.
T110 43501-43691 Epistemic_statement denotes Moreover, based on the extent of divergent evolution within the NiRAN domain it cannot be excluded that the enzyme performs different roles in different nidoviruses (Lehmann et al., 2015a) .
T111 43692-43855 Epistemic_statement denotes The core of the nidovirus RTC likely consists of the RdRp domaincontaining nsp and other viral RNA-binding proteins, like nsp7-10 and nsp13-16 in the case of CoVs.
T112 43856-44135 Epistemic_statement denotes In the infected cell, these subunits assemble into a membrane-bound complex that is associated with a network of modified host membranes, presumably derived from the endoplasmic reticulum (reviewed in (Hagemeijer et al., 2012; Neuman et al., 2014; van der Hoeven et al., 2016) ).
T113 44136-44386 Epistemic_statement denotes An alternative approach to assay nidovirus RNA synthesis in vitro, without the use of purified recombinant proteins, is based on the isolation of the membrane-associated RTCs from infected cells (van Hemert et al., 2008a (van Hemert et al., , 2008b .
T114 44676-44840 Epistemic_statement denotes The synthesis of SARS-CoV and EAV genome and sg mRNAs can be readily reproduced using such a PNS supplemented with [␣-32 P]-CTP, Mg 2+ and an ATP-generating system.
T115 45115-45421 Epistemic_statement denotes Further characterization of this host factor using EAV RTCs showed that it has a mass ranging from 59 to 70 kDa, that it is conserved among animals, but not lower eukaryotes, and that it can be added to inactive RTC preparations in the form of a cytosolic fraction that was extracted from uninfected cells.
T116 45422-45594 Epistemic_statement denotes Presently, the exact nature of this host factor remains unknown (van Hemert et al., 2008a) and it may also not be the only host factor required for nidovirus RNA synthesis.
T117 45595-45939 Epistemic_statement denotes Protein-protein (Pfefferle et al., 2011) and protein-RNA interaction studies, focusing on the 5 and 3 termini of the genome as putative targets (Galan et al., 2009; Spagnolo and Hogue, 2000; Tan et al., 2012) , have suggested that a number of host factors, such as poly(A)-binding protein (PABP), are critical components of the CoV RTC as well.
T118 46297-46486 Epistemic_statement denotes (Decroly et al., 2011; Ma et al., 2015; Zhai et al., 2005) ) have implicated several additional CoV nsps and the nucleocapsid (N) protein as potential viral cofactors of the RNA polymerase.
T119 46487-46793 Epistemic_statement denotes For the AV PRRSV, using different technical approaches, multiple host proteins that appear to interact with nsp9 were identified, although it remains to be studied whether they directly bind to the RdRp domain of the protein and affect its function(s) in RNA synthesis Li et al., 2014a; Liu et al., 2016) .
T120 46794-47044 Epistemic_statement denotes Since the exact role of these (presumed) host and viral co-factors of the nidovirus RNA polymerase remains to be explored, we will now focus on two proteins whose role in nidovirus RNA synthesis has been defined in more detail: AV nsp1 and CoV nsp14.
T121 47045-47240 Epistemic_statement denotes In infected cells, the nidoviral genome and sg mRNAs are all produced with a specific relative abundance and it is poorly understood how the RNA polymerase is controlled to maintain these ratios.
T122 48350-48567 Epistemic_statement denotes This step appears to be primarily directed by TRSs, the short conserved sequence motifs that are present in the genomic template both at the 3 end of the leader sequence and at the 5 end of each of the sg mRNA bodies.
T123 48568-48794 Epistemic_statement denotes When minus strand RNA synthesis has been attenuated at a body TRS, the body TRS complement, which forms the 3 end of the nascent minus strand, can base pair with the genomic leader TRS to direct re-initiation of RNA synthesis.
T124 49153-49501 Epistemic_statement denotes In addition to TRS base pairing, higher-order RNA structure likely co-determines the relative efficiency of different TRSs in nidovirus transcription, which mechanistically resembles the process of copy-choice RNA recombination, as it is thought to commonly occur among +RNA viruses (Pasternak et al., 2001; Sola et al., 2015; Yount et al., 2006) .
T125 49502-49737 Epistemic_statement denotes The above strongly suggests that nidovirus replication and transcription are competing for common factors, like the RNA polymerase, and that the balance between continuous and discontinuous minus strand RNA synthesis must be regulated.
T126 49738-49907 Epistemic_statement denotes In addition to regulatory RNA signals like the TRSs, specific replicase subunits may interact with the RNA polymerase to influence its behaviour on the genomic template.
T127 49908-50046 Epistemic_statement denotes Solid support for this hypothesis was obtained in the EAV model, in particular for the nsp1 "transcription factor" and the nsp10 helicase.
T128 50047-50327 Epistemic_statement denotes Specific mutations in these two subunits can (nearly) completely inactivate transcription (Nedialkova et al., 2010; Seybert et al., 2005; Tijms et al., 2001; van Dinten et al., 1997) , with significant upregulation of genome replication being a prominent and striking side-effect.
T129 50513-50711 Epistemic_statement denotes The Nterminal zinc finger domain of nsp1 is important for this function, but also mutations in other nsp1 domains can strongly influence transcription (Nedialkova et al., 2010; Tijms et al., 2001) .
T130 50712-50939 Epistemic_statement denotes Nsp1 mutagenesis and pseudorevertant analysis provided genetic evidence that a balanced ratio between replication and transcription, and also between individual sg mRNA species, is vital to the virus (Nedialkova et al., 2010) .
T131 50940-50999 Epistemic_statement denotes However, it remains unclear how AV nsp1 interacts with e.g.
T132 51195-51217 Epistemic_statement denotes The protein could e.g.
T133 51373-51529 Epistemic_statement denotes Studies aiming to detect AV nsp1-nsp9 or nsp1-nsp10 interactions have not been successful so far, but it is conceivable that such complexes are short-lived.
T134 51530-51744 Epistemic_statement denotes Clearly, a robust in vitro AV RdRp assay could be a ground-breaking tool to explore attenuation during minus-strand RNA synthesis, TRS base pairing and the role of regulatory protein factors like AV nsp1 and nsp10.
T135 51745-51900 Epistemic_statement denotes The composition of the RNA-synthesizing complexes that other nidoviruses employ for replication and transcription has not yet been studied in great detail.
T136 51901-52072 Epistemic_statement denotes Their much larger genome size than the AV genome clearly provides ample grounds to speculate that they may encode mechanisms and factors that are lacking in the AV system.
T137 52073-52210 Epistemic_statement denotes Still, thus far only a few studies have suggested the existence of CoV "transcription factors" with an impact resembling that of AV nsp1.
T138 52211-52451 Epistemic_statement denotes A point mutation in the helicase (nsp13) of the CoV infectious bronchitis virus was reported to cause a specific block in transcription (Fang et al., 2007) , but this observation has not been followed up in more detail for any CoV thus far.
T139 52709-52897 Epistemic_statement denotes The RdRp complexes engaged in replication and transcription may differ, as transcription-specific regulatory protein factors, like EAV nsp1, have been described (Nedialkova et al., 2010) .
T140 53070-53353 Epistemic_statement denotes Inhibition of GSK-3 was found to reduce the accumulation of MHV genome and longer, but not shorter, sg mRNAs, a result that could be reproduced upon the siRNA-mediated knockdown of the cellular helicase DDX1, which was found to interact with the phosphorylated form of the N protein.
T141 53354-53582 Epistemic_statement denotes Recruitment of DDX1 may play a role in suppressing the attenuation of minus strand RNA synthesis at body TRSs, thus increasing the production of templates for the subsequent synthesis of the longer sg mRNAs and the viral genome.
T142 53779-54058 Epistemic_statement denotes Clearly other differences may exist, but the attenuation of minus strand RNA synthesis to produce subgenome-length templates for mRNA synthesis does appear to be a conserved nidovirus feature, with a clear mechanistic connection to the processivity and other features of the RTC.
T143 54059-54295 Epistemic_statement denotes The discovery that a CoV was the causative agent of the SARS epidemic, inspired a renewed bioinformatics analysis and led to the identification of five previously unknown RNA processing enzymes in the CoV genome (Snijder et al., 2003) .
T144 54662-54938 Epistemic_statement denotes This correlation between genome size and the presence of ExoN triggered the hypothesis that the enzyme may have been acquired to assist the RNA polymerase in achieving sufficient replication fidelity to support the unusually large genome size of the ExoN-encoding nidoviruses.
T145 55108-55315 Epistemic_statement denotes The Mg 2+ -or Mn 2+ -dependent 3 -to-5 exoribonuclease activity of recombinant SARS-CoV nsp14 could indeed be confirmed in vitro on double-stranded and single-stranded RNA templates (Minskaia et al., 2006) .
T146 55344-55656 Epistemic_statement denotes discovered that the in vitro exoribonuclease activity of nsp14 can be stimulated more than 35-fold by addition of nsp10, a small ORF1a-encoded protein that also interacts with nsp16 to enhance that subunit's 2 -O-methyl transferase activity, which is involved in CoV mRNA cap modification (Bouvet et al., 2012) .
T147 55657-55930 Epistemic_statement denotes The preferred substrates of the nsp10+14 exoribonuclease complex in vitro are double-stranded RNAs with or without a 3 -terminal mismatch, while RNA duplexes with longer stretches of mismatching nucleotides were found to be degraded less efficiently (Bouvet et al., 2012) .
T148 55931-56255 Epistemic_statement denotes Experimental support for the hypothesis that the ExoN domain of nsp14 influences the fidelity of CoV RNA synthesis was first obtained using an MHV ExoN knockout mutant (carrying a D89A/E91A double mutation in conserved exoribonuclease motif I (Snijder et al., 2003; Zuo and Deutscher, 2001) ), which was crippled but viable.
T149 56805-57036 Epistemic_statement denotes In line with the hypothesis that ExoN catalyses the excision of 3terminal mismatched nucleotides during RNA synthesis, the MHV ExoN knockout mutant was more sensitive to the mutagenic nucleoside analogue 5-FU (Smith et al., 2013) .
T150 57299-57502 Epistemic_statement denotes A single nsp10 molecule interacts with the ExoN domain of nsp14, which supports the available biochemical data that nsp10 only activates ribonuclease activity (Bouvet et al., 2010; Bouvet et al., 2012) .
T151 57503-57686 Epistemic_statement denotes Despite the presence of two unusual zinc fingers on the surface of the ExoN domain, the core structure of the enzyme is similar to that of members of the DEDD exonuclease superfamily.
T152 57965-58175 Epistemic_statement denotes A direct interaction of SARS-CoV nsp14 with nsp12 was observed in pull-down assays (Imbert et al., 2008) and the protein could also be co-purified with the nsp7+8+12 tripartite complex (Subissi et al., 2014b) .
T153 58288-58434 Epistemic_statement denotes N7-MTase, RdRp and ExoN, suggesting that the nsps involved may cooperate in the large CoV RTC during viral RNA-synthesis (Subissi et al., 2014b) .
T154 58435-58695 Epistemic_statement denotes Nucleotide excision by ExoN presumably occurs during RNA replication, likely following a strand transfer between the nsp12 RdRp domain and the nsp14 ExoN domain after a mismatching nucleotide has been incorporated (Bouvet et al., 2012; Subissi et al., 2014b) .
T155 58931-59236 Epistemic_statement denotes Given the stable viable phenotype of the ExoN domain knockout mutants in MHV and SARS-CoV, it is remarkable that similar mutants could not be recovered for two distantly related CoVs: human CoV-229E (HCoV-229E) and transmissible gastroenteritis virus (TGEV) (Becares et al., 2016; Minskaia et al., 2006) .
T156 59237-59457 Epistemic_statement denotes Although this may be a specific characteristic of the CoV cluster (genus alphacoronavirus) to which they belong, these findings may also reflect the complexity of the functions that nsp14 fulfills during CoV replication.
T157 59458-59826 Epistemic_statement denotes In this respect, it is also noteworthy that ExoN mutations in TGEV, HCoV-229E and MHV have been reported to influence viral replication and transcription (Becares et al., 2016; Eckerle et al., 2007; Minskaia et al., 2006) and that the phenotype of ExoN knockout mutants can be strongly influenced by the innate immune response of the host cell (Becares et al., 2016) .
T158 60825-60937 Epistemic_statement denotes Nucleoside analogues can inhibit viral replication directly when their incorporation leads to chain termination.
T159 60938-61243 Epistemic_statement denotes Alternatively, the analogue can affect viral replication indirectly by inducing so-called 'lethal mutagenesis' when the incorporation of a mutagen into nascent RNAs increases the viral mutation rate, leading to the accumulation of (too many) mutations in the viral genome, and the extinction of the virus.
T160 61244-61468 Epistemic_statement denotes The inhibition of poliovirus replication by treatment with ribavirin (RBV), which is both an adenosine and guanosine analogue, is believed to mainly occur via the latter mechanism (Crotty et al., 2002; Crotty et al., 2001) .
T161 61469-61684 Epistemic_statement denotes The combination of RBV and IFN-␣2a or IFN-␤1a is one of the experimental treatment options for MERS patients, despite disappointing results obtained with RBV treatment during the SARS epidemic in (Lau et al., 2009 .
T162 61685-61940 Epistemic_statement denotes As in SARS patients, high mortality rates in comparison with untreated patients are also reported for MERS patients, but the study outcomes are difficult to compare due to significant differences in study design (for a review, see (Mo and Fisher, 2016) ).
T163 61941-62131 Epistemic_statement denotes In vitro and in small animal models, however, RBV in combination with IFN has been shown to inhibit MERS-CoV infection (Chan et al., 2015; Falzarano et al., 2013a; Falzarano et al., 2013b) .
T164 62132-62507 Epistemic_statement denotes The inhibition of CoV replication in cell culture by RBV alone has been investigated repeatedly for SARS-CoV, MERS-CoV and other CoVs (FCoV, PEDV) and was found to be highly variable (Chen et al., 2004; Chu et al., 2004; Cinatl et al., 2003; Hart et al., 2014; Lee et al., 2015; Morgenstern et al., 2005; Stroher et al., 2004; Tan et al., 2004; Weiss and Oostrom-Ram, 1989) .
T165 62508-62783 Epistemic_statement denotes The results of these cell culture-based studies need to be interpreted with caution as the uptake of RBV by several cell lines, including the frequently used African green monkey kidney (Vero) cells, is known to be inefficient (Ibarra and Pfeiffer, 2009; Shah et al., 2010) .
T166 63259-63442 Epistemic_statement denotes This mechanism of action may also explain the inhibition of AV (PRRSV) replication by RBV treatment, which was not accompanied by an increased mutation frequency (Kim and Lee, 2013) .
T167 63443-63595 Epistemic_statement denotes In summary, it remains questionable if the incorporation of RBV-triphosphate is the mechanism of action for the inhibition of the nidovirus replication.
T168 63596-63888 Epistemic_statement denotes In the case of CoVs, also the nucleotide excision activity of the nsp14 ExoN domain may contribute to the insensitivity of viral RNA synthesis to mutagenic nucleotide analogues, provided that the ExoN domain can detect the subtle chemical differences between these compounds and regular NTPs.
T169 63889-64064 Epistemic_statement denotes In line with this hypothesis, inactivation of MHV and SARS-CoV ExoN activity dramatically increased (up to 300-fold) the sensitivity to the mutagen 5-FU (Smith et al., 2013) .
T170 64065-64339 Epistemic_statement denotes Deep sequencing of RNA produced by wt SARS-CoV in the presence of 5-FU revealed an increase in the frequency of mutagen-associated A-to-G and U-to-C transitions, indicating that 5-FU-monophosphate was incorporated into the viral genome even in the presence of ExoN activity.
T171 64340-64485 Epistemic_statement denotes However, such mutations were more frequently found in the RNA produced under similar conditions by an ExoN knockout mutant (Smith et al., 2013) .
T172 64486-64721 Epistemic_statement denotes The observation that even the wt virus incorporates 5-FU is consistent with the finding that nsp12 RdRp domain mutations (see above) can improve the selectivity of the viral RNA polymerase for 5-FU incorporation (Sexton et al., 2016) .
T173 64722-64923 Epistemic_statement denotes Although the nsp14 ExoN activity may interfere with the effectiveness of using nucleoside analogues to inhibit CoV RNA synthesis, recently three such compounds were reported to inhibit CoV replication.
T174 65794-65957 Epistemic_statement denotes The IVRA, as developed for SARS-CoV and EAV and described above, may prove to be an alternative approach to probe the mechanism of action of polymerase inhibitors.
T175 65958-66176 Epistemic_statement denotes For example, using these assays, Zn 2+ was previously identified as an inhibitor of RNA synthesis for both viruses, presumably by interfering with initiation (EAV) or elongation (SARS-CoV) (te Velthuis et al., 2010b) .
T176 66299-66503 Epistemic_statement denotes However, evidence that any of these targets the CoV polymerase directly is still lacking, as biochemical assays with purified nsp12 and the triphosphate versions of these compounds remain to be performed.
T177 66625-66857 Epistemic_statement denotes Clearly, more details on the structure and function of the CoV polymerase could accelerate the development of this type of anti-CoV drugs, which should definitely be part of our first line of defense against emerging CoV infections.
T178 67037-67248 Epistemic_statement denotes Unraveling how these important RNA viruses replicate and transcribe their genome is crucial for understanding nidovirus biology and the discovery of drugs for the prevention or treatment of nidovirus infections.
T179 67587-67776 Epistemic_statement denotes Possibly because of this complexity, the nidovirus RdRp-encoding subunit possesses unique features, like the presence of the N-terminally linked NiRAN domain with nucleotidylation activity.
T180 67777-67969 Epistemic_statement denotes Overall, however, the nidovirus RTC, which is associated with modified intracellular membranes, is poorly understood nor do we know its exact composition in terms of viral and host components.
T181 68201-68400 Epistemic_statement denotes The addition of other nsps as co-factors (nsp7 and nsp8) in the in vitro CoV polymerase assays appears to offer a promising avenue for further studies and, eventually, assays for inhibitor screening.
T182 68401-68625 Epistemic_statement denotes Besides the further development of robust in vitro polymerase assays, preferable also for the AV polymerase, several key questions remain to be answered, including (i) how do nidovirus RNA polymerases initiate RNA synthesis?
T183 68625-68710 Epistemic_statement denotes ; (ii) how do the ExoN and RdRp domains work together to promote polymerase fidelity?
T184 68770-68861 Epistemic_statement denotes (iv) how is the switch from transcription to replication controlled at the molecular level?
T185 69212-69343 Epistemic_statement denotes In order to successfully produce new infectious virus particles, these events need to be coordinated and must be tightly regulated.
T186 69344-69561 Epistemic_statement denotes Some regulation may be spatial (replication vs translation), other forms of regulation are most likely determined by specific RNA sequences and/or the conformation of the 5 -and 3 -terminal sequences of the viral RNA.
T187 69562-69674 Epistemic_statement denotes How these signals affect the activity of the RNA polymerase and other RTC subunits merits further investigation.
T188 69863-70086 Epistemic_statement denotes This information will deepen our understanding of the evolutionary process that has produced these unusually complex RNA virus genomes, and may aid in finding ways to combat current and future emerging nidovirus infections.