key: cord-0936378-mdgjqtdd
authors: Nelson, Christopher A.; Pekosz, Andrew; Lee, Chung A.; Diamond, Michael S.; Fremont, Daved H.
title: Structure and Intracellular Targeting of the SARS-Coronavirus Orf7a Accessory Protein
date: 2005-01-11
journal: Structure
DOI: 10.1016/j.str.2004.10.010
sha: 65d22c9c8eb2b0cd987ff566f6bc6a1dc7a5211e
doc_id: 936378
cord_uid: mdgjqtdd

The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 Å resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded β sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

compactly folded monomer. We verified the identity of most favored region of the Ramachandran plot and the remaining 5.2% in the additionally allowed region as the orf7a ectodomain fragment by electrospray mass defined by PROCHECK (Laskowski et al., 1993) . There spectrometry (see Experimental Procedures). The obare no residues in the disallowed or generously allowed served mass is consistent with two disulfide bonds in regions. The final model has an R work of 22.3% to 1.8 Å the refolded molecule. The protein runs as a single speresolution, with an R free of 27.5%. cies on native PAGE and is stable in solution at 10 mg/ Our model for the orf7a luminal domain consists of ml over a period of several weeks. seven β strands which form two β sheets, compactly arranged in an Ig-like β sandwich fold ( Figure 1B ). How-Structure of the Orf7a Luminal Domain ever, the precise topology of orf7a is distinctive from We next initiated a structural-genomics-type examinathat of typical Ig-superfamily members ( Figure 1C ). tion of orf7a. We hoped to gain insight into the potential Strand A, instead of running antiparallel to strand B, function of orf7a by investigating the structural relationhas switched sheets and lies parallel to strand G. Like ships between orf7a and other well-characterized promany C1-type Ig domains, both the C# and C$ strands teins. Crystallization screening of the refolded orf7a are absent. In addition, the C and D strands are both protein yielded diffraction-quality hexagonal crystals very short: the C strand is only 4 amino acids in length, belonging to space group P3 1 (a = b = 37.10 Å, c = while the D strand is just 3 residues long. The structure 55.33 Å), which grew over a 3 day period in hanging of orf7a also contains two unusual disulfide bonds condrops to an approximate size of 0.2 × 0.1 × 0.1 mm.

necting the BED and AGFC sheets. Neither occurs in Initial phasing was accomplished by multiple isomorthe typical position occupied by the Ig-superfamily caphous replacement (MIR) using data collected from three nonical disulfide (i.e., connecting the B and F strands). different Pt heavy atom derivatives (Table 1 ). The result-

The first disulfide of orf7a connects the end of strand ing electron density maps were readily interpretable A to the E-F loop, while the second disulfide connects ( Figure 1A ). An initial model, spanning residues 1-67 the short B-C and F-G loops. without a main chain break, was obtained directly from We sought to identify proteins of similar topology by experimental phase using the autobuild feature of ARP/ performing a Dali search (Holm and Sander, 1995 with 6% sequence identity) and the N-terminal domain and immunoprecipitation assays (Table 2A) . To prove that the IgG 2b isotype clone 2E11 was specific for na-of the human interleukin-1 receptor (IL-1R) (PDB ID 1IRA, fragment 1-94, rmsd of 2.0 Å for 46 aligned resi-tive orf7a, immunoprecipitation studies were performed using lysate from SARS-CoV-infected cells. Vero cells dues, with 11% sequence identity). Both structures are considered examples of I-set Ig domains as defined by were infected with SARS-CoV at a multiplicity of infection (moi) of 0.01 for 48 hr in accordance with the rules SCOP (Murzin et al., 1995) . Comparisons of orf7a with 55 additional members of the Ig superfamily identified and regulations of Washington University and the "Laboratory Biosafety Guidelines for Handling and Process-by Dali revealed sequence identities ranging from 2% to 16% for aligned core residues, consistent with our ing Specimens Associated with SARS-CoV" as put forward by the Department of Health and Human Services failure to predict an Ig-like fold from the primary sequence alone. Also, compared with most Ig folds, the Centers for Disease Control and Prevention (CDC). All manipulation of infectious samples took place in a BSL-3 orf7a luminal domain is extremely small, consisting of only 65 amino acids. For comparison, the average biological safety facility. The infected cells were washed, solubilized in 1.0% Triton X-100, and immune com-length of an I-set domain in HOMSTRAD (Mizuguchi et al., 1998) is 98 amino acids. In addition to the short C plexes were recovered on protein A Sepharose. As expected, the 2E11 clone immunoprecipitated a single and D strands, the membrane-distal B-C and F-G loops are also comparatively short. These differences are band of w12 kDa. This protein was identified as orf7a by N-terminal sequencing (Table 2B ). An identical se-best seen in the structure-based alignment of the I-set domains with orf7a ( Figure 1D ). quence was obtained using lysate from 293T cells transfected with an orf7a cDNA. In both cases, the predicted 15 amino acid signal peptide (MKIILFLTLIVFTSC)

In order to characterize the expression and intracellular of orf7a had been removed, presumably by signal peptidase cleavage. These results and the inability of 2E11 trafficking of orf7a, we generated monoclonal antibodies (mAbs) specific for the luminal domain. Recombi-to recognize denatured (boiled-reduced) recombinant orf7a protein on Western blot support the conclusion nant soluble-refolded orf7a protein was used to immunize mice. Solid-phase ELISA identified 24 hybridomas that 2E11 is specific for orf7a in its natively expressed form. The ability of the conformation-dependent 2E11 producing mAb specific for the refolded protein. These were characterized in cell staining, Western blotting, mAb (raised against recombinant, refolded orf7a) to im- tively charged residues proximal to the membrane (Lys103, Arg104, and Lys105 in Figure 1D )

two Lys tail residues were changed to Ala. The orf7a-AA-GFP tail mutant, like the wild-type, displayed only bution, a green fluorescence protein (GFP) tag was fused in-frame to the carboxyl terminus. The fluores-a low level of expression at the plasma membrane of transfected cells ( Figure 3A , lower panels). The majority cent pattern was clearly distinct from GFP alone ( Figure  2K ). The fusion tag also did not alter the observed intra-of the mutant protein colocalized with antibodies directed against the ER resident protein calnexin ( 

). The orf7a-GFP distribution was distinct from that seen for GFP alone (K). marker protein retained in the Golgi, as seen by com-perfamily. This common structural fold occurs in a wide variety of proteins, where it performs a diverse set of paring the colocalization of this chimeric construct with Golgin 97 ( Figure 4B ). However, localization of the CD4/ functions. For example, the fold is found in proteins of the extracellular matrix, muscle proteins, proteins of the orf7a TM tail construct was not as tight as that seen for orf7a alone, suggesting that residues in the orf7a 

What other functions could orf7a serve within the context of the ER/Golgi network? A significant number of viral accessory proteins have been found to be critical for the evasion of host-mediated immunity, interfering with diverse processes, including apoptosis, complement activation, cytokine signaling, and innate and adaptive immune surveillance (Alcami and Koszinowski, 2000; Ploegh, 1998). While many of these proteins act at the plasma membrane or are secreted from infected cells, others have been found to operate within 

At 48 hr postinfection, the cells were washed in 50 mM HEPES (pH expression vector pET-21a. This plasmid was introduced into 7.5), 1 mM EGTA, 1500 µM MgCl 2 , 150 mM NaCl, and 1× complete BL21(DE3)-RIL codon (+) E. coli cells for expression. Recombinant protease inhibitors (Roche), then suspended at 1 × 10 7 cells/ml in protein was recovered as an inclusion body pellet, denatured, rethe same buffer. The cells were lysed with an equal volume of 2.0% duced, and then renatured by rapid dilution in refolding buffer con-Triton X-100 in 150 mM NaCl. After centrifugation, supernatants sisting of 1 M arginine, 100 mM Tris-HCl, 2 mM EDTA, 200 µM were precleared with protein A Sepharose. The anti-orf7a mAb PMSF, 5 mM reduced glutathione, and 500 µM oxidized glutathione 2E11 was added to 10 g/ml and held 60 min at 4°C. Immune comat a final pH of 8.3. After 24 hr, the soluble-refolded protein was plexes were recovered on protein A Sepharose and washed in cold collected over YM10 membrane in a stirred cell concentrator, lysis buffer without protease inhibitors before being heated to 90°C passed through a 0.45 m filter, and subjected to sizing on Superin loading buffer (62.5 mM Tris-HCl [pH 6.0], 2% SDS, 5% 2-merdex 200 using an AKTA-FPLC. The original cDNA clone differed by captoethanol, 500 mM sucrose) for 5 min. After separation on SDSa single nucleotide from the published sequence (C/A at position PAGE, the proteins were transferred to polyvinylidene difluoride 27,360 as numbered in NCBI Accession No. AY278741.1). All of our membrane. The blot was rinsed in distilled water and then methaconstructs maintain the resulting Leu15 to Ile15 replacement. The nol before soaking 2 min in 1.1% w/v Coomassie blue, 40% v/v final recombinant fragment of orf7a spanned amino acid residues methanol/water, and 1% acetic acid. Several changes of 50% M(−3)SC/ELY···EEVQQE81* and contained no extraneous tags. methanol were required to destain the blot. The orf7a region was excised for N-terminal sequencing (Table 2B) . 

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