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CORD-19:ba334a8f734285257878e0df4e64a6131818d0c6 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-26 Sentence denotes Nidovirus RNA polymerases:
T2 27-75 Sentence denotes Complex enzymes handling exceptional RNA genomes
T3 77-85 Sentence denotes Abstract
T4 86-204 Sentence denotes Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales.
T5 205-421 Sentence denotes Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes.
T6 422-683 Sentence denotes Coronaviruses (26-34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps.
T7 684-836 Sentence denotes The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function.
T8 837-1008 Sentence denotes The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses.
T9 1009-1180 Sentence 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).
T10 1181-1385 Sentence 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.
T11 1386-1562 Sentence denotes In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3 -to-5 exoribonuclease activity in nsp14 was implicated in fidelity.
T12 1563-1690 Sentence denotes In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production.
T13 1691-1897 Sentence denotes Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.
T14 1899-2201 Sentence 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.
T15 2202-2706 Sentence 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) .
T16 2707-2974 Sentence denotes Animal CoVs continue to emerge and cause great economic losses, as exemplified by the recent outbreaks of porcine deltacoronavirus and the porcine epidemic diarrhea virus (PEDV) in China and the United States (Choudhury et al., 2016; Weng et al., 2016; Zhang, 2016) .
T17 2975-3234 Sentence denotes Genetically, the nidoviruses constitute a monophyletic group that is characterized by common ancestry of their key replicative enzymes and associated similarities in genome organization and expression ( Fig. 1A ) (de Groot et al., 2012; Lauber et al., 2013) .
T18 3235-3435 Sentence 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) .
T19 3436-3803 Sentence denotes It has been postulated that this size variation reflects a long history of gradual genome expansion, during which the different nidovirus lineages adapted to their specific niches by acquiring a range of novel functions, encoded by domains that were either incorporated as additional genes or integrated into the large nidovirus replicase gene (Lauber et al., 2013) .
T20 3804-4010 Sentence denotes In all nidoviruses, at least two-thirds of the capacity of the polycistronic genome is occupied by the two large open reading frames (ORFs; 1a and 1b) that together constitute the replicase gene (Fig. 1A) .
T21 4011-4219 Sentence denotes Both ORFs are translated directly from the viral genome and briefly overlap where a −1 ribosomal frameshift directs the expression of ORF1b to facilitate the formation of an ORF1abencoded polyprotein (pp1ab).
T22 4220-4581 Sentence denotes Cleavage of the pp1a and pp1ab polyproteins by multiple intrinsic protease activities, in combination with −1 and −2 frameshifting in the nsp2 coding region in most arteriviruses with the exception of equine arteritis virus (EAV), results in the production of 13 to 17 non-structural proteins (nsps) (Fang et al., 2012; Li et al., 2014b; Ziebuhr et al., 2000) .
T23 4582-5057 Sentence denotes The common ancestry of the extremely diverged nidovirus lineages is primarily reflected in the conservation of an array of 'core replicase domains' Lauber et al., 2013; Snijder et al., 2016) , which is composed of two trans-membrane proteins, the viral main protease, and -encoded downstream of the ORF1a/1b frameshift site -the RNA-dependent RNA polymerase (RdRp)-and helicase-containing subunits (Fig. 1B) , with the canonical RdRp domain residing in CoV nsp12 and AV nsp9.
T24 5058-5195 Sentence denotes Nidovirus genomes are thought to have expanded gradually by gene duplication and the acquisition of novel domains (Lauber et al., 2013) .
T25 5196-5410 Sentence 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.
T26 5411-5762 Sentence 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) .
T27 5763-5916 Sentence denotes Consequently, some nidovirus nsps contain a unique set of activities not seen in other +RNA viruses (discussed e.g. in Sections 8 and 11 in more detail).
T28 5917-6312 Sentence denotes Imaging and biochemical characterization of nidovirus nsps have shown that they are targeted to specific virus-induced membrane structures (reviewed in (Hagemeijer et al., 2012; Neuman et al., 2014; van der Hoeven et al., 2016) ) where they assemble into a so-called replication and transcription complex (RTC; see (Neuman et al., 2014; Snijder et al., 2016; Subissi et al., 2014a) for reviews).
T29 6313-6447 Sentence 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.
T30 6448-6715 Sentence denotes In the case of nidoviruses and their polycistronic genomes, RNA synthesis entails not only genome amplification but also the synthesis of a nested set of subgenomic (sg) mRNAs ( Fig. 1A ; reviewed in (Pasternak et al., 2006; Sawicki et al., 2007; Sola et al., 2011) .
T31 6716-6833 Sentence denotes The sg mRNAs serve to make the genes downstream of the nidovirus replicase ORFs 1a and 1b accessible for translation.
T32 6834-7044 Sentence denotes These ORFs encode structural and so-called 'accessory' proteins, which are often dispensable for replication in vitro, but important for e.g. immune evasion and pathogenesis in vivo Weiss and Leibowitz, 2011) .
T33 7045-7220 Sentence 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.
T34 7221-7303 Sentence denotes Each nidoviral sg mRNA is produced from a complementary subgenome-length template.
T35 7304-7571 Sentence 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) .
T36 7572-7901 Sentence 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.
T37 7902-8223 Sentence 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) .
T38 8224-8425 Sentence denotes The same essentially applies to the major cis-acting RNA elements that constitute the initiation sites for minus and plus strand RNA synthesis near the 3 end of the plus and minus strand, respectively.
T39 8426-8652 Sentence denotes Indeed, in both CoV and AV genomes, a number of primary and higher-order structural features have been identified and studied, including sev- GAV, gill-associated virus; note that the EAV genome is drawn to a different scale).
T40 8653-8831 Sentence 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.
T41 8832-8936 Sentence denotes ORFs encoding 'accessory proteins' (in SARS-CoV) or poorly characterized products are depicted in white.
T42 8937-9249 Sentence denotes To illustrate the principle of subgenomic mRNA synthesis, as employed by all nidoviruses, the nested set structure and composition of the mRNAs is summarized for SARS-CoV, with the common 5 leader sequence indicated in red and the translated part of the genome and each of the subgenomic mRNAs depicted in green.
T43 9250-9436 Sentence denotes See main text for more details. (B) Domain organization of the pp1ab replicase polyprotein for the four major nidovirus lineages (note that the AV protein is drawn to a different scale).
T44 9437-9531 Sentence denotes Proteolytic cleavages and non-structural protein numbering are indicated for EAV and SARS-CoV.
T45 9532-9859 Sentence denotes The scheme highlights the conservation of the so-called nidovirus 'core replicase', consisting of the ORF1a-encoded main protease (Mpro) flanked by two transmembrane (TM) domains, followed by the ORF1b-encoded NiRAN nucleotidyl transferase (NT), RNA polymerase (RdRp), zinc binding domain (Z) and superfamily 1 helicase (HEL1).
T46 9860-9977 Sentence denotes Accessory (papain-like) protease domains and their cleavage sites are indicated for EAV and SARS-CoV (P1, P2, PLpro).
T47 9978-10094 Sentence denotes The zinc-finger domain (F) in EAV nsp1 that is crucial for subgenomic mRNA synthesis (see text) is also highlighted.
T48 10095-10503 Sentence denotes The C-terminal part of pp1ab encodes a number of enzymatic domains that are not strictly conserved among all nidovirus lineages: U, endoribonuclease, conserved in vertebrate nidoviruses; EN, exoribonuclease (ExoN) conserved in nidoviruses with genome sizes >20 kb (see text); N7-and 2 -O methyl transferases (N7 and 2O) involved in cap modification (not identified in AVs). eral RNA hairpins and pseudoknots.
T49 10504-10647 Sentence denotes The importance of multiple of these elements is supported by bioinformatics (conservation), biochemical probing, and site-directed mutagenesis.
T50 10648-10948 Sentence denotes For an overview of the structure and function of the RNA elements that have been implicated in CoV or AV replication and transcription, the reader is referred to detailed reviews that have been published elsewhere (Madhugiri et al., 2016; Masters, 2007; Sola et al., 2011; Yang and Leibowitz, 2015) .
T51 10949-11385 Sentence 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. 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) .
T52 11386-11701 Sentence 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.
T53 11702-11874 Sentence 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.
T54 11875-12084 Sentence denotes The ability of the nidovirus RTC to direct the various processes of RNA synthesis outlined above critically relies on the activity of a canonical RdRp domain, which resides in nsp12 in CoVs and in nsp9 in AVs.
T55 12085-12286 Sentence 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.
T56 12287-12502 Sentence denotes Given their pivotal role in viral replication and the efficacy of using polymerase inhibitors to combat other virus infections, AV nsp9 and CoV nsp12 are also considered an important target for rational drug design.
T57 12503-12815 Sentence denotes Unfortunately, these nsps have remained refractory to structural analyses so far, but significant advances in our understanding of these enzymes and their co-factors have been made using bioinformatics, biochemical and molecular virological approaches, in particular for AVs and CoVs, as we will summarize below.
T58 12816-13083 Sentence denotes In all nidoviruses, the RdRp-containing subunit consists of at least two domains ( Fig. 2A) : a nidovirus-specific N-terminal domain is followed by a canonical RdRp domain occupying the C-terminal two-thirds of the protein (Gorbalenya et al., 1989; Xu et al., 2003) .
T59 13084-13337 Sentence 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.
T60 13338-13528 Sentence denotes The domain is now referred to as the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain (Lehmann et al., 2015a) (Fig. 2B) , and it will be discussed in more detail in Section 8.
T61 13529-13755 Sentence 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. 2A ) may turn out to connect the NiRAN and RdRp domains (Lehmann et al., 2015a) .
T62 13756-13828 Sentence denotes RdRp domains include six canonical conserved motifs, named A to F (Figs.
T63 13829-13983 Sentence denotes 2C and 3A ), that are involved in template and substrate recognition and the catalysis of nucleotide condensation (Poch et al., 1989; te Velthuis, 2014) .
T64 13984-14161 Sentence denotes Nucleotide condensation particularly depends on motifs A and C, which assist in coordinating the two metal ions in the active site (Alphonse and Ghose, 2017; te Velthuis, 2014).
T65 14162-14613 Sentence denotes Nidovirus RdRp domains were first identified by sequence comparison with known RdRp domains from other virus groups, revealing an SDD sequence (instead of the usual GDD signature found in other positive-strand RNA virus RdRp domains) as the key residues of motif C (den Boon et al., 1991; Gorbalenya et al., 1989; Snijder et al., 1990 ) (residues 759-761 in SARS-CoV) and D618 and D623 as the conserved aspartate residues of motif A of SARS-CoV nsp12.
T66 14614-14731 Sentence denotes These residues correspond with D445 and D450 in motif A and SDD at positions 559-561 in nsp9 of the AV prototype EAV.
T67 14732-15025 Sentence denotes Two decades later, the mutational analysis of conserved RdRp domain residues confirmed that the catalytic aspartates of motifs A and C are important for RNA polymerase activity and viral RNA synthesis (Ahn et al., 2012; Lehmann et al., 2016; Subissi et al., 2014b; te Velthuis et al., 2010a) .
T68 15026-15212 Sentence denotes In addition to the essential RdRp domain motifs mentioned above, CoV nsp12 has a motif G, which is considered a signature sequence for primer-dependent RNA polymerases Xu et al., 2003) .
T69 15213-15303 Sentence denotes Remarkably, this motif is only partially conserved in the AV nsp9 (Beerens et al., 2007) .
T70 15304-15561 Sentence denotes A structural prediction for the SARS-CoV RdRp domain was published as early as 2003 (Xu et al., 2003) (Fig. 3B ), yet experimental studies to support this model have been greatly hampered by difficulties with attempts to stably express and purify CoV nsp12.
T71 15562-15650 Sentence denotes As a result, the crystal structure of the CoV RNA polymerase remains (Xu et al., 2003) .
T72 15651-15755 Sentence denotes All three models are based on the C-terminal polymerase domain and excluded the N-terminal NiRAN domain.
T73 15756-15796 Sentence denotes All models show an overall similar fold.
T74 15797-15881 Sentence denotes Differences exist in the fingers subdomain and surface loops of the thumb subdomain.
T75 15882-15977 Sentence denotes C) Superposed models of the EAV nsp9 RdRp generated by use of I-TASSER, Swiss-Model and Phyre2.
T76 15978-16078 Sentence denotes Overall, all three models show a very similar fold, with small differences in the fingers subdomain.
T77 16079-16190 Sentence denotes D) Motifs of the polymerase domain of SARS-CoV nsp12 indicated on the structural model 1O5S (Xu et al., 2003) .
T78 16191-16270 Sentence denotes E) Motifs of the polymerase domain of EAV nsp9 that was generated using Phyre2.
T79 16271-16379 Sentence denotes F) Key active site and fidelity residues indicated on the model of polymerase domain of SARS-CoV nsp12 1O5S.
T80 16380-16452 Sentence denotes to be solved and its biochemical characterization in a very early stage.
T81 16453-16530 Sentence denotes Unfortunately, the situation is not much different for the AV RNA polymerase.
T82 16531-16764 Sentence denotes Most of our current understanding of the structural and enzymatic features of the nidovirus RNA polymerase/RTC is based exclusively on studies with SARS-CoV, which was targeted by a number of laboratories following the 2003 outbreak.
T83 16765-16854 Sentence denotes To date, no cryo-EM or crystallographic structures of CoV nsp12 or AV nsp9 are available.
T84 16855-17082 Sentence 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. 3B ) and MHV (Sexton et al., 2016) has been predicted.
T85 17083-17583 Sentence 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. 3B ) revealed a right-handed fold that consists of thumb, palm and fingers subdomains, similar to known crystal structures of RdRp domains or complete RNA polymerases (shown for foot and mouth disease virus (FMDV) in Fig. 3A ).
T86 17584-17741 Sentence denotes The models also predict the presence of a single-stranded (ss) RNA entry channel at the top of the polymerase and an NTP entry channel at the rear (Fig. 3) .
T87 17742-17981 Sentence 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.
T88 17982-18344 Sentence 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) .
T89 18345-18712 Sentence 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. 3C ), in line with earlier sequence analyses suggesting that the overall molecular architecture is conserved among nidovirus RdRp domains (Beerens et al., 2007) .
T90 18713-18952 Sentence denotes In neither the CoV nor the AV RdRp domain model there is evidence for the presence of a primingloop or similar initiation platform, which is typically required for de novo initiation on the 3 terminus of the viral RNA (te Velthuis, 2014) .
T91 18953-19135 Sentence 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.
T92 19136-19270 Sentence denotes In both the CoV nsp12 and EAV nsp9 RdRp domain models, the three substrate channels converge at the beta sheets of the palm subdomain.
T93 19271-19446 Sentence denotes Here the catalytic aspartates of motifs A and C coordinate the catalytic ions that are essential for the nucleotide condensation reaction (te Velthuis, 2014) ( Fig. 3D and E).
T94 19447-19634 Sentence 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.
T95 19635-19879 Sentence denotes Further functional predictions about the CoV RNA polymerase come from an alignment of a Phyre2-based molecular model of MHV nsp12 with the crystal structures of the RdRp domains of coxsackievirus B3 (CVB3) and poliovirus (Sexton et al., 2016) .
T96 19880-20106 Sentence denotes In particular, this study identified V553 and M611 (homologs of SARS-CoV nsp12 residues V557 and M615, Fig. 3F ) as putative equivalents of the CVB3 RdRp residues I176 and I230, which are known to be involved in RdRp fidelity.
T97 20107-20329 Sentence 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) .
T98 20330-20529 Sentence denotes The first published attempts to purify a CoV nsp12 employed an N-terminal GST-fusion with SARS-CoV nsp12, which yielded a recombinant protein with poor solubility and stability (Cheng et al., 2005) .
T99 20530-20890 Sentence denotes Since then, advances have been made to improve the stability and yield of SARS-CoV nsp12 by using codon optimization, different purification tags at the N-or C-terminus, or the addition of an N-terminal, cleavable ubiquitin (ub) fusion partner (in combination with a C-terminal His 6 -tag) (Ahn et al., 2012; Subissi et al., 2014b; te Velthuis et al., 2010a) .
T100 20891-21227 Sentence 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).
T101 21228-21359 Sentence denotes The C-terminal His 6 -tag proved suitable to purify nsp12 from E. coli without a significant effect on the stability of the enzyme.
T102 21360-21688 Sentence 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).
T103 21689-21919 Sentence 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).
T104 21920-22070 Sentence denotes Since motif G, a presumed hallmark of primer-dependent polymerases Xu et al., 2003) , was identified in the nsp12 sequence and structural model (Figs.
T105 22071-22175 Sentence denotes 2A and 3B), it was assumed that a primer would be required for the initiation of RNA synthesis by nsp12.
T106 22176-22406 Sentence denotes Studies from both Cheng et al. and te Velthuis et al. showed that the activity of recombinant SARS-CoV GST-nsp12 or nsp12-His 6 was indeed primer-dependent in the presence of Mg 2+ (Cheng et al., 2005; te Velthuis et al., 2010a) .
T107 22407-22553 Sentence denotes By contrast, Ahn et al. found that recombinant SARS-CoV nsp12 with an N-terminal His 6 -tag (His 6 -nsp12) required high concentrations of Mn 2+ .
T108 22554-22733 Sentence denotes Under these conditions, the enzyme was also able to initiate de novo on homopolymeric templates and a sequence representing the 3 terminus of the viral genome (Ahn et al., 2012) .
T109 22734-22854 Sentence denotes Likewise, His 6 -nsp12 was able to synthesize relatively long RNA products in the presence of Mn 2+ (Ahn et al., 2012) .
T110 22855-22967 Sentence denotes The activity of nsp12-His 6 on templates longer than 20 nucleotides was not tested (te Velthuis et al., 2010a) .
T111 22968-23219 Sentence denotes Curiously, the most recent study on recombinant SARS-CoV nsp12 (containing a C-terminal strep-tag) did not find any primer extension, de novo initiation activity, or binding (using 1 M of nsp12-strep) to a primed RNA template (Subissi et al., 2014b) .
T112 23220-23337 Sentence denotes By contrast, the nsp12-His 6 variant was found to bind RNA with an apparent Kd of ∼0.1 M (te Velthuis et al., 2010a).
T113 23338-23597 Sentence denotes The limited reproducibility of these biochemical observations and the scant support for substantial processivity of nsp12 are in stark contrast with the fact that the CoV RNA polymerase is required to replicate and transcribe a ∼30-kb genome during infection.
T114 23598-23750 Sentence denotes Technical differences between these studies (e.g. regarding the constructs and templates used) may have contributed to the mostly contradictory results.
T115 23751-23962 Sentence 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.
T116 23963-24159 Sentence 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) .
T117 24160-24369 Sentence 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) .
T118 24370-24633 Sentence 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) .
T119 24634-24860 Sentence 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.
T120 24861-25047 Sentence denotes In subsequent biochemical analyses, it was found that the Nterminus of SARS-CoV nsp8 modulates its oligomerization and polymerase activity (te Velthuis et al., 2012; Xiao et al., 2012) .
T121 25048-25323 Sentence denotes Moreover, proteolytic cleavage of an inactive nsp8 containing an N-terminal ubiquitin-and C-terminal His 6 -tag showed that its nucleotide condensation abilities (i.e. de novo synthesis but also primer extension activity) can be activated by the removal of the terminal tags.
T122 25324-25708 Sentence 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.
T123 25709-25902 Sentence 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) .
T124 25903-25978 Sentence denotes The de novo RNA synthesis activity of nsp8 was not addressed in this study.
T125 25979-26189 Sentence 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) .
T126 26190-26356 Sentence 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) .
T127 26357-26553 Sentence denotes In both studies, a conserved lysine (K58 in SARS-CoV) was found to be essential for polymerase activity and crucial for the binding of nsp8 to RNA (Imbert et al., 2006; te Velthuis et al., 2012) .
T128 26554-26817 Sentence denotes Another conserved lysine (K82) and several, among Coronavirinae conserved aspartates, including a D/ExD/E motif with D50 and D52 as key residues in SARS-CoV nsp8, affected nsp8's ability to synthesize RNA in vitro (Imbert et al., 2006; te Velthuis et al., 2012) .
T129 26818-27055 Sentence 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) .
T130 27056-27414 Sentence 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) .
T131 27415-27702 Sentence 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.
T132 27703-27824 Sentence denotes Unfortunately, we therefore have to conclude that the question whether nsp8 functions as primase still remains wide open.
T133 27825-27942 Sentence denotes In the next section, we will elaborate on the role of nsp8 as a cofactor for RNA replication by nsp12 in more detail.
T134 27943-27945 Sentence denotes 6.
T135 27946-28050 Sentence denotes The RNA polymerase activity of nsp12 and the role of nsp8 as co-factor: the nsp7+8+12 tripartite complex
T136 28051-28186 Sentence denotes In 2005, the SARS-CoV 22-kDa nsp8 was shown to form a ringlike complex together with the 12-kDa nsp7 (Zhai et al., 2005) ( Fig. 4A-C) .
T137 28187-28368 Sentence 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. 4A ).
T138 28369-28637 Sentence 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) .
T139 28638-28891 Sentence 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. 4D ) (Xiao et al., 2012) .
T140 28892-29141 Sentence 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) .
T141 29142-29294 Sentence 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.
T142 29295-29456 Sentence denotes Indeed, several nsp7+8 complexes have been observed across a number of experiments and studies (te Velthuis et al., 2012; Xiao et al., 2012; Zhai et al., 2005) .
T143 29457-29709 Sentence 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) .
T144 29710-29847 Sentence denotes In contrast to their oligomeric form, the structure of the individual nsp7 and nsp8 subunits is consistent across the structural studies.
T145 29848-30006 Sentence denotes Nsp7 adopts a globular structure that consists mostly of alpha-helices, both in solution and in the nsp7+8 crystal (Johnson et al., 2010; Zhai et al., 2005) .
T146 30007-30120 Sentence denotes Nsp8 can adopt a golf club-like shape that is present in a 'straight' (nsp8-I) and 'bent' (nsp8-II) conformation.
T147 30121-30302 Sentence denotes Both forms are present in the hexadecameric nsp7+8 complex ( Fig. 4B and C) , but only the 'straight' form of nsp8 is seen in the FCoV nsp7+8 complex (Xiao et al., 2012) (Fig. 4D) .
T148 30303-30469 Sentence 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) .
T149 30470-30874 Sentence 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) .
T150 30875-31016 Sentence denotes In multiple earlier studies, nsp7 and nsp8 were also shown to copurify or interact with nsp12 (Imbert et al., 2008; von Brunn et al., 2007) .
T151 31017-31252 Sentence denotes In 2014, Subissi et al. 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) .
T152 31253-31466 Sentence denotes In the presence of Mg 2+ , the nsp7+8+12 tripartite complex was also able to initiate RNA synthesis de novo on a 339-nt long fragment representing the 3 -terminal part of the viral genome (Subissi et al., 2014b) .
T153 31467-31763 Sentence 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) .
T154 31764-31974 Sentence denotes Subissi et al. also found that the nsp7L8+12strep complex had a higher RNA binding affinity than nsp7L8 or nsp12-strep alone, of which the latter was unable to bind to the RNA template (Subissi et al., 2014b) .
T155 31975-32129 Sentence 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.
T156 32130-32334 Sentence denotes Mutagenesis of SARS-CoV nsp8 within the context of the nsp7+8+12 complex resulted in the identification of residues that are important for the interaction between nsp8 and nsp12 (nsp8 P183 and nsp8 R190).
T157 32335-32528 Sentence denotes As in studies on nsp8 alone and the nsp7+8 complex, residue K58, which is fully conserved among members of the Coronaviridae family, was found to be involved in the binding of the RNA template.
T158 32529-32820 Sentence 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) .
T159 32821-33104 Sentence 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.
T160 33105-33383 Sentence denotes Nsp8 mutants D52A or K82A, which were found to cripple the nsp7+8 activity in vitro (te Velthuis et al., 2012) and viral RNA synthesis in cell culture (Subissi et al., 2014b) , did not affect nsp7+8+12 complex formation or the primer extension activity of the nsp7+8+12 complex.
T161 33384-33624 Sentence denotes This suggests that the RdRp activity of nsp7+8 as observed by te Velthuis et al. 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.
T162 33625-33808 Sentence 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.
T163 33809-34039 Sentence denotes Mutagenesis of D760 in motif C of SARS-CoV nsp12 RdRp domain (Fig. 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.
T164 34040-34145 Sentence denotes In light of the previous biochemical studies on nsp8 and nsp12, it is difficult to interpret this result.
T165 34146-34281 Sentence 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.
T166 34282-34527 Sentence 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.
T167 34528-34759 Sentence 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.
T168 34760-34898 Sentence denotes Clearly, both the role of nsp8 (primase and/or processivity factor?) and the initiation mechanism employed by nsp12 require further study.
T169 34899-35060 Sentence 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.
T170 35061-35150 Sentence denotes The outside of the hexadecamer is predominantly negatively charged (red surface shading).
T171 35151-35309 Sentence denotes B) The SARS-CoV nsp8 crystal structure (pdb 2AHM) resembles a 'golf club' with a long stick at the N-terminus (N) and a head-like shape at the C-terminus (C).
T172 35310-35406 Sentence denotes The nsp8 structure can adopt two conformations, here shaded green (nsp8-I) and orange (nsp8-II).
T173 35407-35542 Sentence denotes C) In the hexadecamer each of the two nsp8 structures is present four times and complemented by eight nsp7 subunits that act as mortar.
T174 35543-35613 Sentence denotes Two orientations of the nsp7 subunit are indicated (orange and green).
T175 35614-35751 Sentence denotes D) The FCOV heterotrimeric nsp7-8 complex (pdb 3ub0) consists of one nsp8 (shaded green) subunit and two nsp7 subunits (orange and blue).
T176 35752-35799 Sentence denotes The nsp8 subunit adopts an nsp8-I conformation.
T177 35800-35905 Sentence denotes To our knowledge, attempts to achieve in vitro activity of an AV RdRp have only been made using EAV nsp9.
T178 35906-36102 Sentence 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) .
T179 36103-36252 Sentence denotes Beerens et al. reported that recombinant EAV nsp9 is capable of de novo initiation of RNA synthesis on homopolymeric templates (poly(C) and poly(U)).
T180 36253-36444 Sentence 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) .
T181 36445-36581 Sentence 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.
T182 36582-36793 Sentence denotes Although no active site mutant was tested in the initial EAV nsp9 study, the de novo initiation on the poly(U) template was repeated in a subsequent study with a wild-type (wt) EAV nsp9 and D445A motif A mutant.
T183 36794-36923 Sentence denotes In these assays, the D445A mutant showed a significantly reduced activity compared to the wt control (te Velthuis et al., 2010b).
T184 36924-37099 Sentence denotes Unfortunately, more recent efforts using the same T7-driven expression construct failed to reproduce these observations regarding the activity of nsp9 (Lehmann et al., 2016) .
T185 37100-37381 Sentence 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) .
T186 37382-37490 Sentence denotes It was thus concluded that contaminating trace amounts of T7 RNA polymerase may have produced these results.
T187 37491-37698 Sentence 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).
T188 37699-37947 Sentence denotes Unfortunately, this recombinant version of nsp9 showed no de novo polymerase activity, while the primer-dependent and terminal transferase activities were insensitive to replacement of the conserved motif A and C aspartates (Lehmann et al., 2016) .
T189 37948-38200 Sentence 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.
T190 38201-38424 Sentence 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) .
T191 38425-38620 Sentence 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.
T192 38621-38885 Sentence 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.
T193 38886-39099 Sentence denotes In a recent study that combined extensive bioinformatics analysis, biochemical studies and reverse genetics, a nucleotidylation activity was assigned to this domain, which was named NiRAN (Lehmann et al., 2015a) .
T194 39100-39383 Sentence 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.
T195 39384-39539 Sentence denotes Despite limited sequence conservation and significant size differences, from ∼220 residues in AV to over 300 residues in CoV, three motifs were identified:
T196 39540-39666 Sentence denotes 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).
T197 39667-39769 Sentence denotes Together these contain only seven residues that are absolutely conserved among nidoviruses (Fig. 2B ).
T198 39770-39998 Sentence denotes There is a marked size difference in the spacing between NiRAN domain motifs B N and C N when comparing AV with all other nidovirus lineages: these motifs are adjacent in AV, but separated by 40-60 residues in other nidoviruses.
T199 39999-40245 Sentence 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) .
T200 40246-40347 Sentence denotes Nucleotidylation activity was shown for recombinant EAV nsp9, expressed in and purified from E. coli.
T201 40348-40613 Sentence denotes Incubation of the protein with [␣-32 P]GTP or [␣-32 P]UTP, but not [␥-32 P]-labelled nucleotides, resulted in labelling of nsp9 itself, which was attributed to the covalent binding of monophosphate nucleotides while releasing pyrophosphate (Lehmann et al., 2015a) .
T202 40614-40720 Sentence denotes This reaction was Mn 2+dependent and displayed a 5-fold stronger affinity for UTP over GTP as a substrate.
T203 40721-40840 Sentence denotes Most likely a phosphoamide bond is formed between the nucleotide and a conserved lysine in motif A N (K54 in EAV nsp9).
T204 40841-41076 Sentence 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.
T205 41077-41252 Sentence 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.
T206 41253-41562 Sentence denotes For both EAV nsp9 and SARS-CoV nsp12, it was shown that the NiRAN domain is essential for viral replication: reverse engineered mutants with substitutions in the motifs of the NiRAN domain were either non-viable or crippled, in which case they mostly reverted back to the wt sequence (Lehmann et al., 2015a) .
T207 41563-41730 Sentence 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.
T208 41731-41821 Sentence denotes In addition, the role of this activity in the nidovirus replicative cycle remains unknown.
T209 41822-42050 Sentence 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) .
T210 42051-42248 Sentence 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) .
T211 42249-42485 Sentence 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).
T212 42486-42661 Sentence 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.
T213 42662-42818 Sentence denotes So far, three possible functions have been proposed for the nucleotidylation activity of the NiRAN domain in nidoviral replication (Lehmann et al., 2015a) .
T214 42819-43047 Sentence 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) .
T215 43048-43297 Sentence 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) .
T216 43298-43500 Sentence denotes Each of these hypotheses has been discussed in more detail by Lehmann et al. (2015a) , but since all three require additional assumptions about nidovirus RNA synthesis they are still highly speculative.
T217 43501-43691 Sentence 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) .
T218 43692-43855 Sentence 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.
T219 43856-44135 Sentence 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) ).
T220 44136-44386 Sentence 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 .
T221 44387-44526 Sentence denotes To prepare RTCs for this in vitro RNA synthesis assay (IVRA), infected cells are mechanically disrupted and fractionated by centrifugation.
T222 44527-44675 Sentence denotes Low speed centrifugation steps yield a post-nuclear supernatant (PNS), from which nuclei, large debris and remaining intact cells have been removed.
T223 44676-44840 Sentence 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.
T224 44841-45114 Sentence denotes Additional fractionation of the PNS, during which the RTCcontaining membranes were separated from the cytosol fraction by a high-force centrifugation step, revealed that a soluble host factor is required for CoV and AV in vitro RNA synthesis (van Hemert et al., 2008a, b) .
T225 45115-45421 Sentence 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.
T226 45422-45594 Sentence 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.
T227 45595-45939 Sentence 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.
T228 45940-46486 Sentence denotes Similarly, yeast 2-hybrid (Y2H) and mammalian 2-hybrid (M2H) experiments (Imbert et al., 2008; Pan et al., 2008; Prentice et al., 2004; von Brunn et al., 2007) , pull down assays or co-purifications (e.g. (Brockway et al., 2003; Imbert et al., 2008; Subissi et al., 2014b; Sutton et al., 2004; von Brunn et al., 2007) ) and co-crystallization studies (e.g. (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.
T229 46487-46793 Sentence 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) .
T230 46794-47044 Sentence 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.
T231 47045-47240 Sentence 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.
T232 47241-47468 Sentence denotes As outlined in the introduction, each sg mRNA is produced from a subgenome-length complement (Fig. 5) , which derives from a process of discontinuous minus strand RNA synthesis (Sawicki and Sawicki, 1995; Sethna et al., 1989) .
T233 47469-47769 Sentence denotes Whereas continuous minus strand synthesis produces the full-length template (antigenome) for genome replication, discontinuous minus-stranded RNA synthesis yields a nested set of shorter templates for sg mRNA production (reviewed in (Pasternak et al., 2006; Sawicki et al., 2007; Sola et al., 2011) .
T234 47770-48049 Sentence denotes In most nidovirus families, including AVs and CoVs, the sg mRNAs consist of sequences that are non-contiguous in the viral genome: a common 5 'leader' sequence is attached to different 'body' segments representing a variable part of the 3proximal region of the genome (Fig. 1A) .
T235 48050-48164 Sentence denotes The joining of leaderand body-encoding sequences occurs during discontinuous minus strand RNA synthesis (Fig. 5) .
T236 48165-48349 Sentence denotes Following initiation of minus strand RNA synthesis, which invariably occurs at the genomic 3 end, a unique mechanism of 'polymerase jumping' forms the basis for leader-to-body joining.
T237 48350-48567 Sentence 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.
T238 48568-48794 Sentence 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.
T239 48795-48917 Sentence denotes Subsequently, the subgenome-length minus strand is completed by addition of the complement of the genomic leader sequence.
T240 48918-49152 Sentence denotes The importance of (−)TRS-(+)TRS base pairing was probed and confirmed extensively by site-directed mutagenesis and reverse genetics for both AVs and CoVs (reviewed in (Pasternak et al., 2006; Sawicki et al., 2007; Sola et al., 2011) .
T241 49153-49501 Sentence 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) .
T1 49502-49737 Sentence 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.
T2 49738-49907 Sentence 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.
T3 49908-50046 Sentence denotes Solid support for this hypothesis was obtained in the EAV model, in particular for the nsp1 "transcription factor" and the nsp10 helicase.
T4 50047-50327 Sentence 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.
T5 50328-50512 Sentence denotes EAV nsp1 was concluded to control the levels at which the genomelength and different subgenome-length minus strand templates accumulate in the infected cell (Nedialkova et al., 2010) .
T6 50513-50711 Sentence 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) .
T7 50712-50939 Sentence 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) .
T8 50940-51194 Sentence denotes However, it remains unclear how AV nsp1 interacts with e.g. the network of TRS signals, the viral RNA polymerase (AV nsp9), or the helicase (AV nsp10), which has also been implicated in transcriptional control (Deng et al., 2014; Lehmann et al., 2015b) .
T9 51195-51372 Sentence denotes The protein could e.g. modulate RTC stalling at body TRSs, nascent minus strand transfer to the leader TRS or reinitiation of RNA synthesis following (−)TRS-(+)TRS base pairing.
T10 51373-51529 Sentence 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.
T11 51530-51744 Sentence 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.
T12 51745-51900 Sentence denotes The composition of the RNA-synthesizing complexes that other nidoviruses employ for replication and transcription has not yet been studied in great detail.
T13 51901-52072 Sentence 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.
T14 52073-52210 Sentence denotes Still, thus far only a few studies have suggested the existence of CoV "transcription factors" with an impact resembling that of AV nsp1.
T15 52211-52451 Sentence 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.
T16 52452-52642 Sentence denotes More recently, N Guided by a base-pairing interaction between the −B and +L sequences, RNA synthesis is resumed to add the anti-leader sequence to each nascent subgenome-length minus strand.
T17 52643-52708 Sentence denotes Subsequently, the latter serves as template to produce a sg mRNA.
T18 52709-52897 Sentence 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) .
T19 52898-52928 Sentence denotes For further details, see text.
T20 52929-53069 Sentence denotes Adapted from protein phosphorylation by the host kinase GSK-3 was reported to affect the relative abundance of MHV mRNAs in infected cells .
T21 53070-53353 Sentence 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.
T22 53354-53582 Sentence 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.
T23 53583-53778 Sentence denotes A mechanistic difference among different nidovirus lineages that has emerged is the lack of a common leader sequence in the sg mRNAs of some groups (Cowley et al., 2002; van Vliet et al., 2002) .
T24 53779-54058 Sentence 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.
T25 54059-54295 Sentence 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) .
T26 54296-54471 Sentence denotes One of them is a 3 -to-5 exoribonuclease (ExoN) residing in the N-terminal domain of nsp14, which also contains a C-terminal N7-methyl transferase domain (Chen et al., 2009) .
T27 54472-54661 Sentence denotes This ExoN domain is present in all nidoviruses with a genome size above 20 kb (corona-, toro-, roni-, and mesoniviruses, but not arteriviruses; (Snijder et al., 2003; Lauber et al., 2012) .
T28 54662-54938 Sentence 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.
T29 54939-55107 Sentence denotes By analogy with the 3 -to-5 exonuclease domain of DNA polymerases, it was speculated that ExoN provides some kind of error correction mechanism (Snijder et al., 2003) .
T30 55108-55315 Sentence 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) .
T31 55316-55656 Sentence denotes Subsequently, Bouvet et al. 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) .
T32 55657-55930 Sentence 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) .
T33 55931-56255 Sentence 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.
T34 56256-56426 Sentence denotes This mutant displayed a mutator phenotype and -compared to the wt control -it accumulated ∼15-fold more neutral mutations during serial passaging (Eckerle et al., 2007) .
T35 56427-56652 Sentence denotes This mutator phenotype was confirmed for SARS-CoV, with the corresponding ExoN mutant accumulating about 20 times more mutations, which were randomly distributed across the genome (Eckerle et al., 2010; Graham et al., 2012) .
T36 56653-56804 Sentence denotes Although these two ExoN knockout mutants replicate more slowly, they have proven to be remarkably stable (Eckerle et al., 2010; Eckerle et al., 2007) .
T37 56805-57036 Sentence 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) .
T38 57037-57298 Sentence denotes The recently solved crystal structure of the SARS-CoV nsp10+14 complex revealed that nsp14 folds into two separate domains, with residues 1-287 and 288-527 forming the N-terminal ExoN and Cterminal N7-methyl transferase domains, respectively (Ma et al., 2015) .
T39 57299-57502 Sentence 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) .
T40 57503-57686 Sentence 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.
T41 57687-57964 Sentence denotes CoV ExoN contains five catalytic residues (D90, E92, E191, H268 and D273 in SARS-CoV), and the enzyme was classified into the subgroup of the so-called DEDDh exonucleases (Ma et al., 2015) , with E191 replacing a D residue that is conserved in other members of the superfamily.
T42 57965-58175 Sentence 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) .
T43 58176-58434 Sentence denotes This multi-nsp complex was found capable of performing all the known enzymatic activities of its subunits, i.e. 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) .
T44 58435-58695 Sentence 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) .
T45 58696-58930 Sentence denotes The structural and biochemical information obtained for nsp14 thus far provides an excellent basis to explore the function(s) and control of the unique nidoviral ExoN enzyme, and in particular its interactions with the RNA polymerase.
T46 58931-59236 Sentence 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) .
T47 59237-59457 Sentence 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.
T48 59458-59826 Sentence 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) .
T49 59827-59961 Sentence denotes The targeting of viral polymerases has been successfully applied as a therapeutic strategy to counter a number of DNA and RNA viruses.
T50 59962-60196 Sentence denotes For RNA viruses, which generally evolve resistance mutations easily due to their high mutation frequency, polymerase inhibitors are commonly administered as part of a cocktail with drugs that target other viral or even host functions.
T51 60197-60467 Sentence denotes Such combination therapies have now become the basis for the successful treatment of infections with human immunodeficiency virus and hepatitis C virus (Banerjee and Reddy, 2016; Majumdar et al., 2016; Nakamura et al., 2016; Poorolajal et al., 2016; Zeng et al., 2013) .
T52 60468-60824 Sentence denotes The development of RNA polymerase inhibitors involves either the (structure-based) design of nucleoside analogues that are recognized by the polymerase and incorporated into the viral RNA genome or the screening for or designing of small molecules that inhibit nucleotide synthesis by binding to the RNA polymerase and prevent it from functioning properly.
T53 60825-60937 Sentence denotes Nucleoside analogues can inhibit viral replication directly when their incorporation leads to chain termination.
T54 60938-61243 Sentence 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.
T55 61244-61468 Sentence 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) .
T56 61469-61684 Sentence 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 .
T57 61685-61940 Sentence 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) ).
T58 61941-62131 Sentence 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) .
T59 62132-62507 Sentence 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) .
T60 62508-62783 Sentence 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) .
T61 62784-63005 Sentence denotes Furthermore, other mechanisms of virus inhibition by RBV have been reported that do not involve the direct targeting of the virus by incorporation of triphosphorylated RBV into nascent RNA (Beaucourt and Vignuzzi, 2014) .
T62 63006-63258 Sentence denotes Recently it was shown that decreased CoV replication mainly results from inhibition of inosine-5 -monophosphate dehydrogenase (IMPDH) by ribavirin monophosphate and the subsequent lowering of cellular GTP pools (Kim and Lee, 2013; Smith et al., 2014) .
T63 63259-63442 Sentence 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) .
T64 63443-63595 Sentence denotes In summary, it remains questionable if the incorporation of RBV-triphosphate is the mechanism of action for the inhibition of the nidovirus replication.
T65 63596-63888 Sentence 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.
T66 63889-64064 Sentence 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) .
T67 64065-64339 Sentence 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.
T68 64340-64485 Sentence denotes However, such mutations were more frequently found in the RNA produced under similar conditions by an ExoN knockout mutant (Smith et al., 2013) .
T69 64486-64721 Sentence 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) .
T70 64722-64923 Sentence 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.
T71 64924-65090 Sentence denotes Two of these were originally identified as filovirus RdRp inhibitors, but were found to display broad-spectrum activity against a range of RNA viruses including CoVs.
T72 65091-65452 Sentence denotes The adenosine analogue BCX4430 inhibits CoV-induced cell death with an EC 50 (half maximal effective concentration) of 58 and 68 M for MERS-and SARS-CoV, respectively (Warren et al., 2014) , whereas GS-5734, an adenosine monophosphate analogue, proved to be strikingly efficient at inhibiting MERS-CoV replication with an EC 50 of 0.34 M (Warren et al., 2016) .
T73 65453-65793 Sentence denotes The third compound with anti-CoV activity is a so-called flexible purine base analogue (fleximer), combined with the acyclic sugar moiety of acyclovir, which was capable of inhibiting HCoV-NL63 and MERS-CoV (but not SARS-CoV) replication with EC 50 values of 9 and 23-25 M, respectively, while acyclovir was inactive (Peters et al., 2015) .
T74 65794-65957 Sentence 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.
T75 65958-66176 Sentence 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) .
T76 66177-66298 Sentence denotes In conclusion, a number of nucleoside analogues have been reported to be able to inhibit CoV replication in cell culture.
T77 66299-66503 Sentence 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.
T78 66504-66624 Sentence denotes In addition, none of these inhibitors has been tested in animal experiments or moved forward to preclinical development.
T79 66625-66857 Sentence 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.
T80 66858-67036 Sentence denotes The order Nidovirales includes a number of important human and animal pathogens, some of which have caused considerable societal unrest and economic damage over the past decades.
T81 67037-67248 Sentence 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.
T82 67249-67385 Sentence denotes Dissecting nidovirus RNA synthesis means unraveling the structure and function of the viral RTC and its core enzyme, the RNA polymerase.
T83 67386-67586 Sentence denotes Nidovirus RTCs must handle and replicate exceptionally large RNA genomes and secure the production of a set of sg mRNAs, which are needed for the expression of their structural and accessory proteins.
T84 67587-67776 Sentence 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.
T85 67777-67969 Sentence 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.
T86 67970-68200 Sentence denotes So far, attempts to develop in vitro assays using purified recombinant polymerases have produced largely contradictory results for both EAV and SARS-CoV, the only two nidoviruses for which experimental data is currently available.
T87 68201-68400 Sentence 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.
T88 68401-68920 Sentence 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?; (ii) how do the ExoN and RdRp domains work together to promote polymerase fidelity?; (iii) what is the function of the conserved NiRAN domain? (iv) how is the switch from transcription to replication controlled at the molecular level?; and (v) what is the composition and structure of the RTC?
T89 68921-69211 Sentence denotes In addition, three steps in the nidovirus replication cycle require the viral genome as a template: the translation of the replicase gene, the synthesis of the antigenome (template for replication) and the production of a set of subgenome-length minus strands (templates for transcription).
T90 69212-69343 Sentence denotes In order to successfully produce new infectious virus particles, these events need to be coordinated and must be tightly regulated.
T91 69344-69561 Sentence 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.
T92 69562-69674 Sentence denotes How these signals affect the activity of the RNA polymerase and other RTC subunits merits further investigation.
T93 69675-69862 Sentence denotes A full understanding of the mechanisms underlying the faithful replication of the nidovirus genome will prove valuable given the unique position nidoviruses occupy in the RNA virus world.
T94 69863-70086 Sentence 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.