Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 3₁₀-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.The coronavirus replication cycle begins with the translation of the 29-kb positive-strand genomic RNA to produce two large polyprotein species (pp1a and pp1ab), which are subsequently cleaved to produce 15 or possibly 16 nonstructural proteins (nsp''s) (11). Among these, nsp3 is the largest nsp and also the largest coronavirus protein. nsp3 is a glycosylated (16, 22), multidomain (36, 51), integral membrane protein (38). All known coronaviruses encode a homologue of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp3, and sequence analysis suggests that at least some functions of nsp3 may be found in all members of the order Nidovirales (11). Hallmarks of the coronavirus nsp3 proteins include one or two papain-like proteinase domains (3, 12, 16, 31, 56, 62), one to three histone H2A-like macrodomains which may bind RNA or RNA-like substrates (5, 9, 48, 54, 55), and a carboxyl-terminal Y domain of unknown function (13). An extensive bioinformatics analysis of the coronavirus replicase proteins by Snijder et al. (51) provided detailed annotations of the then-recently sequenced SARS-CoV genome (35, 47), including the identification of a domain unique to SARS-CoV and the prediction of the ADP-ribose-1″-phosphatase (ADRP) activity of the X domain (since shown to be one of the macrodomains).Only limited information is so far available regarding the ways in which the functions of nsp3 are involved in the coronavirus replication cycle. Some functions of nsp3 appear to be directed toward protein; e.g., the nsp3 proteinase domain cleaves the amino-terminal two or three nsp''s from the polyprotein and has deubiquitinating activity (4, 6, 14, 30, 53, 60). Most homologues of the most conserved macrodomain of nsp3 appear to possess ADRP activity (9, 34, 41-43, 48, 59) and may act on protein-conjugated poly(ADP-ribose); however, this function appears to be dispensable for replication (10, 42) and may not be conserved in all coronaviruses (41). The potential involvement of nsp3 in RNA replication is suggested by the presence of several RNA-binding domains (5, 36, 49, 54, 55). nsp3 has been identified in convoluted membrane structures that are also associated with other replicase proteins and that have been shown to be involved in viral RNA synthesis (16, 24, 52), and nsp3 papain-like proteinase activity is essential for replication (14, 62). Other conserved structural features of nsp3 include two ubiquitin-like domains (UB1 and UB2) (45, 49). We have also recently reported that nsp3 is a structural protein, since it was identified as a minor component of purified SARS-CoV preparations, although it is not known whether nsp3 is directly involved in virogenesis or is incidentally incorporated due to protein-protein or protein-RNA interactions (36).A nucleic acid-binding region (NAB) is located within the polypeptide segment of residues 1035 to 1203 of nsp3. The NAB is expected to be located in the cytoplasm, along with the papain-like protease, ADRP, a region unique to SARS-CoV (the SARS-CoV unique domain [SUD]), and nsp3a, since both the N and C termini of nsp3 were shown previously to be cytoplasmic (38). Two hydrophobic segments are membrane spanning (38), and the NAB is located roughly 200 residues in the N-terminal direction from the first membrane-spanning segment. This paper presents the next step in the structural coverage of nsp3, with the determination of the NAB structure. The structural studies included nuclear magnetic resonance (NMR) characterization of two constructs, an nsp3 construct comprising residues 1035 to 1181 [nsp3(1035-1181)] and nsp3(1066-1203), and complete NMR structure determination for the construct nsp3(1066-1181) (see Fig. ​Fig.8).8). The structural data were then used as a platform from which to investigate the nature of the previously reported single-stranded RNA (ssRNA)-binding activity of the NAB (36). Since no three-dimensional (3D) structures for the corresponding domains in other group II coronaviruses are known and since the SARS-CoV NAB has only very-low-level sequence identity to other proteins, such data could not readily be derived from comparisons with structurally and functionally characterized homologues.Open in a separate windowFIG. 8.Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. {"type":"entrez-protein","attrs":{"text":"AAP41036","term_id":"30795144","term_text":"AAP41036"}}AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. {"type":"entrez-protein","attrs":{"text":"ABN10901","term_id":"124389468","term_text":"ABN10901"}}ABN10901), BtCoV-HKU9-1 (accession no. {"type":"entrez-protein","attrs":{"text":"P0C6T6","term_id":"190360129","term_text":"P0C6T6"}}P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. {"type":"entrez-protein","attrs":{"text":"ABD75496","term_id":"89515407","term_text":"ABD75496"}}ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).     

Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35, 71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (∼10%) mortality rate (69). The ∼30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5′ capped and 3′ polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5′ and 3′ untranslated regions (UTRs). These include two in the 5′ UTR required for BCoV defective interfering (DI) RNA replication (Fig. ​(Fig.1A)1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI region in BCoV (15) has been reanalyzed along with the homologous region in MHV and is now described as comprising SL1 and SL2 (Fig. ​(Fig.1A),1A), of which SL2 has been shown to be a cis-replication structure in the context of the MHV genome (38). In the 3′ UTR, two higher-order cis-replication structures have been identified that function in both DI RNA and the MHV genome. These are a 5′-proximal bulged SL and adjacent pseudoknot that potentially act together as a unit (23, 27, 28, 72) and a 3′-proximal octamer-associated bulged SL (39, 76) (Fig. ​(Fig.1A).1A). In addition, the 5′-terminal 65-nucleotide (nt) leader and the 3′-terminal poly(A) tail have been shown to be cis-replication signals for BCoV DI RNA (15, 60).Open in a separate windowFIG. 1.RNA structures in the BCoV genome tested for nsp1 binding. (A) BCoV 5′-terminal and 3′-terminal cis-acting RNA SL structures and flanking sequences identified for BCoV DI RNA replication. Regions of the genome are identified and SL cis-replication elements are identified schematically. Open boxes at nt 100 and 211 identify AUG start codons for the short upstream ORF and ORF 1, respectively. A closed box at nt 124 identifies the UAG stop codon for the short upstream ORF. Shown below the SL structures are the RNA segments used as ³²P-labeled probes in the gel shift assays. BSL-PK, bulged SL-pseudoknot; 8mer-BSL, octamer-associated bulged SL. (B) Gel shift assays for probes when used with purified nsp1. Protein-RNA complexes identifying a shifted probe are labeled C.In CoVs, the 5′-proximal open reading frame (ORF) of ∼20 kb (called ORF 1) comprising the 5′ two-thirds of the genome is translated to overlapping polyproteins of ∼500 and ∼700 kDa, named pp1a and pp1ab (41). pp1ab is formed by a −1 ribosomal frameshift event at the ORF1a-ORF1b junction during translation (41). pp1a and pp1ab are proteolytically processed into potentially 16 nonstructural protein (nsp) end products or partial end products that are proposed to function together as the replicase (24). ORF 1a encodes nsps 1 to 11 which include papain-like proteases (nsp3), a 3C-like main protease (nsp5), membrane-anchoring proteins (nsps 4 and 6), a potential primase (nsp8), and RNA-binding proteins (nsp 7/nsp 8 complex and nsps 9 and 10) of imprecisely understood function (19, 20, 24, 25, 29, 43, 49, 77). ORF 1b encodes nsps 12 to 16 which function as an RNA-dependent RNA polymerase, a helicase, an exonuclease, an endonuclease, and a 2′-O-methyltransferase, respectively (6, 17, 24, 44). 3′ Proximal genomic ORFs encoding structural and accessory proteins are translated from a 3′-nested set of subgenomic mRNAs (sgmRNAs) (41).The N-terminal ORF 1a protein, nsp1, in the case of BCoV and MHV is also named p28 to identify the cleaved 28-kDa product (18). The precise role of nsp1 in virus replication has not been determined, but it is known that a sequence encoding an N-proximal nsp1 region in MHV (nt 255 to 369 in the 738-nt coding sequence) cannot be deleted from the genome without loss of productive infection (10). nsp1 also directly binds nsp7 and nsp10 (11) and by confocal microscopy is found associated with the membranous replication complex (10, 66) and virus assembly sites (11). The amino acid sequence of nsp1 is poorly conserved among CoVs, indicating that it may be a protein that interacts with cellular components (1, 58). In the absence of other viral proteins, MHV nsp1 induces general host mRNA degradation (79) and cell cycle arrest (16). The SARS-CoV nsp1 homolog, a 20-kDa protein, has been reported to cause mRNA degradation (30, 45), inhibition of host protein synthesis (30, 45, 70), inhibition of interferon signaling (70, 79), and cytokine dysregulation in lung cells (36).In this study, we examine the RNA-binding properties of BCoV nsp1 with the hypothesis that it is a potential regulator of translation or replication through its binding of SLVI mapping within its coding region. The rationale for this hypothesis stems from five observations. (i) In the BCoV DI RNA, the 5′-terminal one-third (approximately) of the nsp1 cistron and the entire nucleocapsid (N) protein cistron together comprise the single contiguous ORF in the DI RNA, and most of both coding regions appear required for DI RNA replication (15). (ii) The partial nsp1 cistron in the DI RNA must be translated in cis for DI RNA replication in helper virus-infected cells (12, 14). (iii) A similar part of the nsp1 cistron is found in the genome of all characterized naturally occurring group 1 and 2 CoV DI RNAs described to date (7, 8). (iv) A cis-acting SL named SLVI is found within the partial nsp1 cistron in the BCoV DI RNA (12). (v) Translation, which involves a 5′→3′ transit of ribosomes, and negative-strand synthesis, which involves a 3′→5′ transit of the RNA-dependent RNA polymerase, cannot simultaneously occur on the same molecule with a single ORF (4, 31). Thus, to enable genome replication an inhibition of translation at least early in infection for cytoplasmically replicating positive-strand RNA viruses is required (4, 5, 22, 32). Mechanisms of translation inhibition have been described for the Qβ viral genome, wherein the viral replicase autoregulates translation by binding an intracistronic cis-replication element (32), and for the polio virus genome, wherein genome circularization inhibits the early translation step (5, 22). Therefore, since nsp1 is synthesized early and also contains an intracistronic cis-replication element, we postulated that it is autoregulatory with RNA binding properties.Here, we do the following: (i) demonstrate by mutagenesis analysis that the 72-nt SLV, mapping immediately upstream of SLVI and within the partial nsp1 cistron, is also a cis-acting DI RNA replication element; (ii) show by gel shift and UV cross-linking analyses that there is likely no binding of an intracellular viral protein to SLV and SLVI (SLV-VI), but there is binding of unidentified cellular proteins of ∼60 and 100 kDa; and (iii) show by gel shift analysis that recombinant nsp1 purified from Escherichia coli does not bind SLV-VI but does bind SLs I to IV in the 5′ UTR and also the 3′-terminal bulged SL in the 3′ UTR, suggesting a possible regulatory role at these sites. Notably, specific binding with ∼2.5 μM affinity of nsp1 to SLIII and its flanking regions in the 5′ UTR was observed. Additionally, we show that, under conditions that would express nsp1 from a DI RNA-encoded sgmRNA, DI RNA levels are greatly reduced; viral RNA species levels, however, are reduced only slightly, and this reduction is transient. These results together indicate that nsp1 is an RNA-binding protein that may function as a regulator of viral translation or replication but not through its binding of cis-acting SLs V and VI within its own cistron.     

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Is Required for Optimal Replication

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several structural proteins, including the spike, nucleocapsid, membrane, and envelope proteins (13). Integrated between and within these structural proteins are eight accessory proteins (6, 8, 10, 15, 16, 18, 21-27). Our laboratory showed previously that one of these SARS-CoV-specific accessory proteins, encoded by ORF6, showed a clearly recognizable phenotype when introduced into a heterologous attenuated murine coronavirus, mouse hepatitis virus (MHV) strain J2.2-V-1 (rJ2.2.6). rJ2.2.6 grew more rapidly and to higher titers in tissue culture cells and in the murine central nervous system than control viruses, and the presence of p6 increased mortality in mice from 10 to 20% to 80% (7, 19, 20). However, the absence of p6 did not diminish SARS-CoV growth in tissue culture cells when cells were infected with 1 PFU/cell (31). In addition to a role in enhancing virus replication, when expressed in the context of a SARS-CoV infection or by transfection, p6 blocked interferon (IFN)-induced STAT1 nuclear translocation by retention of the nuclear import adaptor molecule karyopherin alpha 2 in the cytoplasm, indicating a role in thwarting innate immune effectors (5, 11). In contrast, p6 did not significantly diminish IFN sensitivity when expressed in the context of rJ2.2 (20).The results described above were puzzling, because p6 seemed to be required for the optimal replication of a heterologous coronavirus but not for that of SARS-CoV. Thus, the objective of this study was to determine whether p6 could enhance SARS-CoV replication in tissue culture cells under any conditions. For this purpose, we examined its function by comparing the growth of a recombinant SARS-CoV (rSARS-CoV) in which p6 was deleted (rSARS-CoVΔ6) with that of wild-type rSARS-CoV at a range of multiplicities of infection (MOIs). Normal mice infected with SARS-CoV readily cleared the infection, making it difficult to detect a role for p6 in vivo. However, mice that are transgenic for expression of the human receptor angiotensin-converting enzyme 2 (hACE2) are exquisitely sensitive to infection with SARS-CoV and are useful for identifying an in vivo role for p6 (14).     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.     

A Single Tyrosine in the Severe Acute Respiratory Syndrome Coronavirus Membrane Protein Cytoplasmic Tail Is Important for Efficient Interaction with Spike Protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y₁₉₅, was important for S-M interaction. When Y₁₉₅ was mutated to alanine, M_Y195A no longer retained S intracellularly at the Golgi. Unlike wild-type M, M_Y195A did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y₁₉₅ also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y₁₉₅ is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.Coronaviruses are enveloped positive-strand RNA viruses that infect a wide variety of mammalian and avian species. These viruses generally cause mild disease in humans and are one major cause of the common cold (34). However, severe acute respiratory syndrome coronavirus (SARS-CoV), a novel human coronavirus which emerged in the Guangdong province in China in 2002 (30, 48), caused a widespread pandemic. SARS-CoV caused severe disease with a mortality rate of approximately 10%, the highest for any human coronavirus to date (62). The phylogeny and group classification of SARS-CoV remain controversial (17), but it is widely accepted to be a distant member of group 2. While SARS-CoV is no longer a major health threat, understanding the basic biology of this human pathogen remains important.Coronaviruses encode three major envelope proteins in addition to various nonstructural and accessory proteins. The envelope protein (E) is the least abundant structural protein in the virion envelope, although it is expressed at robust levels during infection (21). E plays an essential role in assembly for some but not all coronaviruses (31-33, 45) and may also be a viroporin (reviewed in reference 21). The spike glycoprotein (S) is the second most abundant protein in the envelope. S determines host cell tropism, binds the host receptor, and is responsible for virus-cell, as well as cell-cell, fusion (15). The S protein is a type I membrane protein with a large, heavily glycosylated luminal domain and a short cytoplasmic tail that has been shown to be palmitoylated in some coronaviruses (47, 58). The membrane protein (M) is the most abundant protein in the virion envelope and acts as a scaffold for virus assembly. M has three transmembrane domains, a long cytoplasmic tail, and a short glycosylated luminal domain (reviewed in reference 21). Unlike many enveloped viruses, coronaviruses assemble at and bud into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and exit the infected cell by exocytosis (29). In order to accomplish this, the envelope proteins must be targeted to the budding compartment for assembly.For most coronaviruses, the E and M proteins localize in the Golgi region near the budding site independently of other viral structural proteins. We have previously shown for infectious bronchitis virus (IBV) E protein that the cytoplasmic tail contains Golgi targeting information (9). IBV M contains Golgi targeting information in its first transmembrane domain (57), while the transmembrane domains and cytoplasmic tail of mouse hepatitis virus (MHV) M appear to play a role in Golgi targeting (1, 36). Some coronavirus S proteins contain targeting information in their cytoplasmic tails; however, some do not (38, 39, 52, 63). Since S proteins can escape to the cell surface when highly expressed, S may rely on lateral interactions with other viral envelope proteins to localize to the budding site and be incorporated into newly assembling virions.In line with its role in virus assembly, M is necessary for virus-like particle (VLP) formation (3, 10, 26, 40, 55, 59). M has been shown to interact with itself to form homo-oligomers (12). In addition, M interacts with E, S, and the viral nucleocapsid and is essential for virion assembly (reviewed in reference 21). Lateral interactions between the coronavirus envelope proteins are critical for efficient virus assembly. The interaction of S and M has been studied for MHV, and the cytoplasmic tail of each protein is important for interaction (16, 44). Specifically, deletion of an amphipathic region in the MHV M cytoplasmic tail abrogates efficient interaction with MHV S (11). The S and M proteins of IBV, bovine coronavirus, feline infectious peritonitis virus, and SARS-CoV have been shown to interact; however, information about the specific regions that are important for interaction remains elusive (16, 22, 26, 42, 64). Due to the presence of several accessory proteins in the virion envelope (23-25, 28, 51, 53), it is possible that the requirements for SARS-CoV S and M interaction could be different from those of previously studied coronaviruses.In earlier work, we reported that SARS-CoV M retains SARS-CoV S intracellularly at the Golgi region when both proteins are expressed exogenously (39). We also demonstrated that the SARS-CoV S cytoplasmic tail interacts with in vitro-transcribed and -translated SARS-CoV M (39). Here, we show that the SARS-CoV M cytoplasmic tail is necessary for specific retention of SARS-CoV S at the Golgi region. We found a critical tyrosine residue at position 195 to be important for retaining SARS-CoV S Golgi membranes when coexpressed with M. When Y₁₉₅ was mutated to alanine, the mutant protein, M_Y195A, did not reduce the amount of SARS-CoV S at the plasma membrane or reduce the extent of S carbohydrate processing as well as wild-type SARS-CoV M does. Additionally, mutation of Y₁₉₅ in SARS-CoV M disrupted the S-M interaction in vitro. Thus, Y₁₉₅ is likely to play a critical role in the assembly of infectious SARS-CoV.     

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca²⁺-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8, 14, 18), where it activates the complement system or acts directly as an opsonin (30, 40, 52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (8, 14, 18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (13, 37, 67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (29, 42, 72, 73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (29, 73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (42).In this study, retroviral particles pseudotyped with SARS-S and in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.     

Toll-Like Receptor 4 Deficiency Increases Disease and Mortality after Mouse Hepatitis Virus Type 1 Infection of Susceptible C3H Mice

Severe acute respiratory syndrome (SARS) is characterized by substantial acute pulmonary inflammation with a high mortality rate. Despite the identification of SARS coronavirus (SARS-CoV) as the etiologic agent of SARS, a thorough understanding of the underlying disease pathogenesis has been hampered by the lack of a suitable animal model that recapitulates the human disease. Intranasal (i.n.) infection of A/J mice with the CoV mouse hepatitis virus strain 1 (MHV-1) induces an acute respiratory disease with a high lethality rate that shares several pathological similarities with SARS-CoV infection in humans. In this study, we examined virus replication and the character of pulmonary inflammation induced by MHV-1 infection in susceptible (A/J, C3H/HeJ, and BALB/c) and resistant (C57BL/6) strains of mice. Virus replication and distribution did not correlate with the relative susceptibilities of A/J, BALB/c, C3H/HeJ, and C57BL/6 mice. In order to further define the role of the host genetic background in influencing susceptibility to MHV-1-induced disease, we examined 14 different inbred mouse strains. BALB.B and BALB/c mice exhibited MHV-1-induced weight loss, whereas all other strains of H-2^b and H-2^d mice did not show any signs of disease following MHV-1 infection. H-2^k mice demonstrated moderate susceptibility, with C3H/HeJ mice exhibiting the most severe disease. C3H/HeJ mice harbor a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts TLR4 signaling. C3H/HeJ mice exhibit enhanced morbidity and mortality following i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.Severe acute respiratory syndrome (SARS) is a disease that was initially observed in 2002 and led to approximately 8,000 affected individuals in multiple countries with over 700 deaths (1, 24, 47, 48). The causative agent of SARS was subsequently identified as a novel coronavirus (CoV) termed SARS-CoV (8, 17, 22, 27, 32, 37). Although SARS-CoV infections following the initial outbreak in 2002 and 2003 have been limited primarily to laboratory personnel, the identification of an animal reservoir for the virus raises concern about the potential for future outbreaks (25).The pathogenesis of SARS has been difficult to study, in part because no animal model is able to fully recapitulate the morbidity and mortality observed in infected humans (35). Infection of a number of inbred mouse strains, including BALB/c, C57BL/6, and 129S, with primary human isolates of SARS-CoV results in the replication of the virus within the lung tissue without the subsequent development of readily apparent clinical disease (11, 16, 41). Infection of aged BALB/c mice results in clinically apparent disease that more closely mimics some aspects of SARS in humans (36). However, immune responses in aged mice are known to be altered (5, 15), and thus, the mechanisms that control the induction of disease may differ between adult and aged mice. Recent work has demonstrated that serial passage of SARS-CoV in mice results in a mouse adaptation that leads to more profound replication of the virus in the lung (28, 34). However, the time to death from this mouse-adapted SARS-CoV is 3 to 5 days, which is much more rapid than the time to mortality observed in fatal cases of SARS in humans.Phylogenetic analysis has revealed that SARS-CoV is most closely related to group 2 CoVs, which include the mouse hepatitis virus (MHV) family (39). Thus, information gathered by infection of mice with closely related members of the group 2 CoVs may further contribute to our understanding of SARS-CoV pathogenesis in humans. While many strains of MHV induce primarily hepatic and central nervous system diseases (6, 7, 12, 18, 21, 23, 40), a recent study demonstrated that intranasal (i.n.) infection of A/J mice with MHV type 1 (MHV-1) induces pulmonary injury that shares several pathological characteristics with SARS-CoV infection of humans (2, 3, 9, 29, 43).In the current study, we examined the relationship between MHV-1 replication in the lungs and the severity of disease in four inbred strains of mice: A/J, BALB/c, C57BL/6, and C3H/HeJ. Our results demonstrate that MHV-1 replicates to similar levels in the lung in each of these inbred strains of mice regardless of their relative levels of susceptibility, as measured by weight loss and clinical illness. Both A/J and C3H/HeJ mice exhibited enhanced weight loss and clinical illness following i.n. MHV-1 infection compared to BALB/c and C57BL/6 mice. Analysis of many different inbred mouse strains confirmed A/J and C3H/HeJ mice as the most susceptible to i.n. MHV-1 infection. Interestingly, C3H/HeJ mice harboring a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts its normal function exhibited greatly increased morbidity and mortality after i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

The Direct Binding of Mrc1, a Checkpoint Mediator,to Mcm6, a Replication Helicase,Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.Progression of the DNA replication machinery along chromosomes is a complex process. Replication forks pause occasionally when they encounter genomic regions that are difficult to replicate, such as highly transcribed regions, tRNA genes, and regions with specialized chromatin structure, like centromeric and heterochromatic regions (17). Replication forks also stall when treated with chemicals like methyl methanesulfonate (MMS), which causes DNA damage, or hydroxyurea (HU), which limits the cellular concentration of the deoxynucleoside triphosphate pool (17). Because de novo assembly and programming of the replisome do not occur after the onset of S phase (18), DNA replication forks must be protected from replicative stresses. The DNA replication checkpoint constitutes a surveillance mechanism for S-phase progression that safeguards replication forks from various replicative stresses (22, 38, 40), and malfunction of this checkpoint leads to chromosome instability and cancer development in higher organisms (4, 9).The Saccharomyces cerevisiae DNA replication checkpoint mediator Mrc1 is functionally conserved and is involved directly in DNA replication as a component of the replisome (1, 8, 16, 19, 29, 30). Mrc1, together with Tof1 and Csm3, is required for forming a replication pausing complex when the fork is exposed to replicative stress by HU (16). The pausing complex subsequently triggers events leading to DNA replication checkpoint activation and hence stable replicative arrest. A sensor kinase complex, Mec1-Ddc2 (ATR-ATRIP homolog of higher eukaryotes), is then recruited to the complex (14, 16). Mec1-Ddc2-mediated phosphorylation of Mrc1 activates the pausing complex, and phosphorylated Mrc1 likely recruits Rad53 (a putative homolog of CHK2 of higher eukaryotes), which is then activated via phosphorylation by Mec1-Ddc2 (1, 16, 20, 30). Activated Rad53 subsequently elicits a stress responses, i.e., stabilization of replication forks, induction of repair genes, and suppression of late-firing origins (24). It remains unclear, however, whether DNA replication checkpoint activation is induced in response to DNA damage by MMS, a reagent commonly used to study the DNA replication stress response. Several lines of evidence have suggested that MMS-induced damage is also sensed directly by the replication machinery (38, 40).Although biochemical and genetic interaction data have placed Mrc1 at the center of the replication checkpoint signal transduction cascade, its molecular function remains largely unknown. The proteins Mrc1, Tof1, and Csm3 associate with the Mcm complex (8, 27), a heterohexameric DNA helicase consisting of Mcm2 to Mcm7 proteins which unwinds the parental DNA duplex to allow replisome progression (3, 12, 18, 31, 32, 35). The Mcm complex associates with a specific set of regulatory proteins at forks to form replisome progression complexes (8). In addition to Mcm, Tof1, Csm3, and Mrc1, replisome progression complexes include factors such as Cdc45 and the GINS complex that are also required for fork progression (13, 26, 31, 32, 39). Claspin, a putative Xenopus laevis homolog of Mrc1, is also reported to associate with Cdc45, DNA polymerase ɛ (Polɛ), replication protein A, and two of the replication factor C complexes in aphidicolin-treated Xenopus egg extracts (19). Recently, Mrc1 was reported to interact directly with Polɛ (23).The aim of this study was to provide mechanistic insight into Mrc1 function in the DNA replication checkpoint. For this purpose, it was essential to identify, among all the essential proteins in the replication machinery, a specific protein that interacts with Mrc1 and to examine the role of this interaction in the DNA replication checkpoint. We found that Mrc1 interacts with Mcm6 directly and specifically. When the interaction between Mrc1 and Mcm6 was impaired, cells no longer activated the DNA replication checkpoint in response to MMS-induced replicative stress. Interestingly and unexpectedly, this interaction was not required for DNA replication checkpoint activation in response to HU-induced replicative stress. Our results provide the first mechanistic evidence that cells use separate mechanisms to transmit replicative stresses caused by MMS and HU for DNA replication checkpoint activation.