2016 Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicati

2016 Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicati

Accepted Manuscript Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles Benjamin W. Neuman PII: S0166-3542(16)30263-7 DOI: 10.1016/j.antiviral.2016.10.005 Reference: AVR 3916 To appear in: Antiviral Research Received Date: 6 May 2016 Revised Date: 23 August 2016 Accepted Date: 12 October 2016 Please cite this article as: Neuman, B.W., Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles, Antiviral Research (2016), doi: 10.1016/j.antiviral.2016.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 1 Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles Benjamin W. Neuman1,2,3 1University of Reading, School of Biological Sciences, University of Reading, RG6 6AJ,6AH United Kingdom 2College of STEM, Texas A&M University-Texarkana, Texarkana TX 75503, USA 3Correspondence to [email protected] [email protected] MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 2 Abstract Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 3 Introduction The order Nidovirales includes several families of large RNA viruses, arranged from longest to shortest genome as the Coronaviridae, Roniviridae, Mesoniviridae and Arteriviridae. The Coronaviridae currently contains the Torovirinae and Coronavirinae lineages, though analysis of recently reported divergent toro-like viruses suggests that the Torovirinae may be better represented as an independent family in the Nidovirales (Stenglein et al., 2014). The Coronavirinae currently contains four genera – Alpha- and Betacoronavirus that infect mammals, and Gamma- and Deltacoronavirus that infect birds and mammals. Members of the Nidovirales infect metazoan hosts, have several replicative genes in common (Lauber et al., 2013), express their structural genes via subgenomic mRNAs which usually join sequences from both genomic termini (Sawicki et al., 2007), express their replicases as polyproteins via a ribosomal frameshift, and replicate in association with viral transmembrane proteins on intracellular paired membranes (reviewed in (V’Kovski et al., 2015)). Coronavirus growth is accompanied by a variety of intracellular membrane rearrangements, as illustrated for the coronavirus Mouse hepatitis virus (MHV) in Fig. 1. Regions of these paired-membrane structures have been given a variety of names in the literature, including double-membrane vesicles (DMVs), convoluted membranes, spherules, zippered endoplasmic reticulum, but it is not clear whether the different parts of the organelle have different functions. Furthermore, recent studies (Al-Mulla et al., 2014; Maier et al., 2016) suggest that there may be considerable plasticity and overlap among coronavirus paired membrane replicative structures. For this reason, it seems preferable to break with past practice of focussing on double-membrane vesicles in particular, to consider the double-membrane organelle (DMO) as a whole. The term DMO encompasses DMVs as well as all of the other associated paired-membrane structures like convoluted membranes and spherules that make up the viral replicative organelle. The term DMO will be used throughout this review except when specifically referring to the DMV component of the replicative organelle. This review summarizes what we know about coronavirus DMOs, and what bioinformatics can tell us about DMO-making proteins. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 4 Origin of DMO Membranes Membrane-bound replicative organelles are a widespread but not universal intracellular feature associated with positive-strand RNA virus replication (Neuman et al., 2014a). Coronaviruses form a double-membrane organelle (DMO) derived from the endoplasmic reticulum (ER) that contains viral RNA and replicase proteins (Deming et al., 2007; Hagemeijer et al., 2010; Oostra et al., 2007; Reggiori et al., 2010; Shi et al., 1999; van der Meer et al., 1999). In the DMO, two lipid bilayers are held at a constant distance of about 20 nm (Angelini et al., 2013). Electron microscopy and tomography studies have revealed that the replicative organelles of coronaviruses and the related arteriviruses are drawn from a repertoire of paired-membranes, including open-ended spherules, closed double-membrane vesicles, and both planar and convoluted paired membranes (Knoops et al., 2012; Knoops et al., 2008; Maier et al., 2013). Nonstructural proteins nsp3, nsp4 and nsp6 are required to form structures similar to the DMOs observed in SARS coronavirus (SARS-CoV) infected cells (Angelini et al., 2013). And since protease domains of nsp5 are required to release nsp4 and nsp6 from the polyprotein precursor, the region from nsp3 to nsp6 can collectively be thought of as the DMO-forming apparatus of coronaviruses. Phylogenetic analysis and comparison of domain architecture can be taken as evidence of homology of the coronavirus DMO-making proteins across the Nidovirales (Fig. 2). The formation of paired membranes probably involves interactions on both sides of the membrane, and perhaps within the membrane, and a few of these interactions have been confirmed biochemically. SARS-CoV nsp3-nsp3 interactions have been detected in cells by yeast two-hybridization (Pan et al., 2008) and GST pulldown (Imbert et al., 2008), and in purified protein by perfluorooctanoic acid polyacrylamide gel electrophoresis (Neuman et al., 2008). While SARS-CoV nsp4-nsp4 interactions were not found in yeast-two hybrid or mammalian two-hybrid screens (Pan et al., 2008; von Brunn et al., 2007) studies with MHV did detect nsp4-nsp4 interactions by Venus reporter fluorescence (Hagemeijer et al., 2011). To date, homotypic interactions have not been demonstrated for nsp6 despite several attempts (Imbert et al., 2008; Pan et MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 5 al., 2008; von Brunn et al., 2007). Heterotypic interactions between coronavirus nsp3 and nsp4 have been observed by mammalian two-hybridization (Pan et al., 2008) and Venus reporter fluorescence (Hagemeijer et al., 2011), although because of differences in the parts of nsp3 that were used, these may actually represent two distinct modes of interaction between nsp3 and nsp4. Nsp4-nsp6 interaction has been demonstrated by Venus reporter fluorescence (Hagemeijer et al., 2011) and indirectly because co- expression of nsp4 abrogates vesicle accumulation due to expression of nsp6 (Angelini et al., 2013). Each of these proteins also potentially interacts with host proteins, which may have a downstream effect on pathogenesis (Pfefferle et al., 2011). Coexpression of nsp3 and nsp4 produces large areas of paired membranes, apparently arranged in parallel tubes (Angelini et al., 2013; Hagemeijer et al., 2014). In terms of topology, nsp3-4 membrane pairing involves linking opposite sides of the ER across the ER lumen, so it is likely that the luminal domains of both proteins are involved in this interaction. Membrane pairing would not necessarily need to involve high-affinity interactions, as demonstrated by a study where paired membranes were induced by low-affinity interactions between membrane-linked green fluorescent protein (Snapp et al., 2003). The minimal requirements for DMO-like membrane pairing appear to be the C-terminal region of nsp3 that contains both transmembrane regions and the luminal ectodomain (Hagemeijer et al., 2014), and at least the N-terminal region of nsp4 including the first three transmembrane regions of nsp4 (Sparks et al., 2007). Mutations in the glycosylated luminal domain of nsp4 result in either non-recoverable virus or less consistent membrane pairing (Gadlage et al., 2010). While the final transmembrane C- terminal cytosolic domain of nsp4 is dispensible for coronavirus replication (Sparks et al., 2007), it is not clear whether partial deletion of nsp4 affects DMO structure. These observations together suggest that coronavirus membranes most likely pair through heterotypic interactions involving the luminal domains of nsp3 and nsp4, though interactions between cytosolic domains that lead to nsp3 and nsp4 clustering may also be important for membrane pairing. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 6 The ER is the likely source of coronavirus DMO membranes, which may be obtained by co-opting the ER-associated degradation (ERAD) tuning pathway, a cellular degradation pathway that is responsible for the turnover of misfolded proteins in the ER (Reggiori et al., 2010). The ERAD tuning pathway is modulated by stress-inducible positive regulators of protein disposal such as EDEM1 (ER degradation-enhancing alpha mannosidase-like 1) and OS-9 (osteosarcoma amplified 9), which assist in transporting misfolded proteins into the cytosol for proteasomal degradation. Under physiological conditions, low concentrations of EDEM1 and OS-9 are maintained in the ER lumen in order to avoid premature degradation of proteins that are in the process of folding (Calì et al., 2008). In this case, EDEM1 and OS-9 are selectively confined by interacting with the transmembrane-anchored cargo receptor SEL1L (suppressor of lin-12-like) and later released from the ER lumen in small short-lived vesicles, called EDEMosomes, which rapidly fuse with the endolysosomal compartments (Bernasconi et al., 2012). In infected cells, viral double-stranded RNA colocalizes with EDEM1, OS-9, SEL1L and LC3-I, which is recruited to autophagosomes. Moreover, replication of MHV, which does not require an intact autophagy pathway, is impaired upon knockdown of LC3 or SEL1L (Bernasconi et al., 2012). Taken together, this is evidence that MHV exploits the ERAD- tuning machinery to establish DMOs for replication. A summary of nsp3-6 interactions and induced membrane rearrangements is shown in Fig. 3. Nsp3 Coronavirus nsp3 is a large multidomain protein ranging from around 1450 amino acid residues in Deltacoronavirus (Woo et al., 2012) to nearly 2100 amino acid residues in the unpublished Hipposideros pratti betacoronavirus-Zhejiang2013 (GenBank accession NC_025217). Most nsp3s are predicted to be ~200 kDa, and are cleaved from the polyprotein 1a or 1ab papain-like proteases (PLpro) that are encoded within nsp3. To make it easier to discuss specific parts of such a large protein in terms of both structure and function, we previously published a domain-level annotation of nsp3 (Neuman et al., 2008), which we have updated for this review (Fig. 4). MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 7 Based on phylogenetic analysis of nidovirus nsp3 homologues, results from previously published studies (Gorbalenya et al., 2006; Ratia et al., 2006; Saikatendu et al., 2005; Serrano et al., 2007b; Thiel et al., 2003; Ziebuhr et al., 2001) and de novo domain prediction software (Jaroszewski et al., 2005), we estimate that the full repertoire of sequenced coronavirus nsp3 genes encodes 18 domains, with individual viruses having 10-16 domains each. Several of these domains are duplicated, including two ubiquitin- like domains Ub1 and Ub2, two PLpro, and three macrodomains (Mac1, Mac2 and Mac3). Ten of these domains form the core of coronavirus nsp3, and are found in every currently known coronavirus, including both ubiquitin-like domains, the second PLpro, the first macrodomain Mac1, a hypervariable region consisting of mostly acidic residues, and a region including the transmembrane regions TM1 and TM2, nsp3 ectodomain (3Ecto), a nidovirus-conserved domain of unknown function (Y) and a region predicted to contain two structural domains which are only found in coronaviruses (CoV-Y). Six of the ten domains conserved in all coronaviruses are also found in other members of the Nidovirales, with evidence that the region from TM1 to Y is present in all nidoviruses except the two families that infect arthropods, namely the Roniviridae and Mesoniviridae (V’Kovski et al., 2015). The ectodomain of nsp3, 3Ecto, is glycosylated in SARS-CoV at positions 1431 and 1434 (Harcourt et al., 2004) and the corresponding region of MHV (Kanjanahaluethai et al., 2007), and is predicted to be located on the luminal side of the membrane. Each copy of nsp3 is predicted span the membrane twice, placing the first 1395 residues of SARS-CoV nsp3 and the last 377 residues (Y and CoV-Y) on the cytosolic face of the membrane. Notably, the regions immediately before TM1 and after TM2, which would both have a cytosolic membrane topology are highly hydrophobic, and may serve to link the pre- and post-transmembrane regions of nsp3. Nsp3 (Ub1 to PL1pro) The N-terminal domain of all coronavirus nsp3 proteins containing Ub1, a hypervariable region (HVR) and PL1pro appears poorly conserved at first glance, showing less than 20% average amino acid identity between members of different coronavirus genera MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 8 (Fig. 5), but secondary structure prediction (JPred; (Drozdetskiy et al., 2015)) suggests that the Ub1 and PL1pro domains adopt a conserved fold in all coronaviruses (data not shown). The NMR structure of the residues 1-112 of SARS-CoV nsp3 exhibits a globular ubiquitin-like fold with two additional helices which make the overall structure of the Ub1 domain somewhat more elongated than other ubiquitin-like proteins (Serrano et al., 2007b). In contrast, the following HVR was shown to be structurally disordered for SARS-CoV (Serrano et al., 2007b) and is dispensable for replication in MHV (Hurst et al., 2013). While the function of Ub1 has only been investigated in MHV, this domain has been found to play an essential role in initiating viral RNA synthesis, where it interacts with the viral nucleoprotein (N; (Hurst et al., 2013; Hurst-Hess et al., 2015)). This was demonstrated in experiments that attempted to delete nsp3 domains, or substitute with the corresponding domains from other coronaviruses. The interaction of Ub1 with N could effectively tether nsp3 to viral RNA during the replication process, but further experimentation is needed to better understand how the nsp3-N interaction leads to more efficient viral RNA synthesis. Additionally, Ub1 has an extra alpha helix and a 310 helix that is unusual for ubiquitin folds in general (Serrano et al., 2007a). Among the closest structural matches to SARS-CoV Ub1 is one of the ubiquitin-like domains of ISG15, an interferon-induced protein constitutively present in higher eukaryotes. This has led to speculation that Ub1 may be involved in modulating the effects of intracellular immunity in a manner analogous to the immunomodulatory decoys of poxviruses (Johnston and McFadden, 2003). Some viruses have developed a mechanism to avoid the expression of ISG15. For example, Influenza B virus blocks its expression by means of NS1 protein in order to overcome the immune response. The PL2pro domain of SARS-CoV nsp3 also recognizes and cleaves ISG15 (Lindner et al., 2007), which could potentially modulate the intracellular response to infection (Morales et al., 2015). However, the comparison with ISG15 remains speculative because an immunomodulatory function for Ub1 has not yet been demonstrated experimentally. It is known that ISG15 is able to inhibit virus MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 9 replication by abrogating nuclear processing of unspliced viral RNA precursors. However, some viruses have developed a mechanism to avoid the expression of ISG15. For example, influenza B virus blocks its expression by means of NS1 protein in order to overcome the immune response. It is possible that the PL2pro domain of nsp3 may also bind ISG15 and modulate the intracellular response to infection (Morales et al., 2015). However, the comparison with ISG15 remains speculative because an immunomodulatory function for Ub1 has not yet been demonstrated experimentally. When SARS-CoV Ub1 was expressed in E. coli, it was found to bind tightly to a small RNA fragment that mass spectrometry analysis revealed to be consistent with the sequence GAUA or GUAA (Serrano et al., 2007a). While matching sequences can be found throughout the SARS-CoV, none are prominently located in regions of the 5’-UTR or 3’-UTR that are known to contain sequences essential for recognition of viral RNA by components of the replicase. The functional significance of RNA-binding by Ub1 therefore remains unknown, but may complement the Ub1-N interaction. A papain-like protease domain (PL1pro) follows the HVR domain in some coronaviruses, but is absent in SARS-CoV and Middle east respiratory syndrome coronavirus (MERS- CoV). Where present, PL1pro generally cleaves at the N-terminal boundary of nsp3, but in viruses that have only one PLpro, this cleavage is carried out by PL2pro (Hilgenfeld, 2014). A transcription factor-like zinc finger is conserved in all complete coronavirus PLpro domains (Culver et al., 1993), which was taken as an early indication that nsp3 might be involved in coronavirus RNA synthesis. This hypothesis is supported by a report in which the Equine arteritis virus nonstructural protein 1, which is structurally and enzymatically similar to coronavirus PL1pro (Sun et al., 2009), was shown to be indispensable for viral subgenomic mRNA synthesis (Phizicky and Greer, 1993). Some strains of Infectious bronchitis virus and Hipposideros pratti betacoronavirus- Zhejiang2013 contain only partial papain-like protease domains that are lacking one of the two catalytic domains (Fig. 2). It is not clear whether these domains are able to interact with the missing domains from PL2pro and remain functional, or whether these

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