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JOURNAL OF VIROLOGY July 2008 p 7212 7222 Vol 82 No 14 0022 538X 08 08 00H110010 doi 10 1128 JVI 02406 07 Copyright 2008 American Society for Microbiology All Rights Reserved Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Hepatitis Virus Infections by More than One Mechanism H17188 Snawar Hussain 1 Stanley Perlman 2 and Thomas M Gallagher 1 Department of Microbiology and Immunology Loyola University Medical Center Maywood Illinois 1 and Interdisciplinary Program in Immunology Department of Microbiology University of Iowa Iowa City Iowa 2 Received 7 November 2007 Accepted 25 April 2008 The severe acute respiratory syndrome coronavirus SARS CoV encodes numerous accessory proteins whose importance in the natural infection process is currently unclear One of these accessory proteins is set apart by its function in the context of a related murine hepatitis virus MHV infection SARS CoV protein 6 increases MHV neurovirulence and accelerates MHV infection kinetics in tissue culture Protein 6 also blocks nuclear import of macromolecules from the cytoplasm a process known to involve its C terminal residues interacting with cellular importins In this study protein 6 was expressed from plasmid DNAs and accumu lated in cells prior to infection by wild type MHV Output of MHV progeny was significantly increased by preexisting protein 6 Protein 6 with C terminal deletion mutations no longer interfered with nuclear import processes but still retained much of the capacity to augment MHV infections However some virus growth enhancing activity could be ascribed to the C terminal end of protein 6 To determine whether this augmen tation provided by the C terminus was derived from interference with nuclear import we evaluated the virus modulating effects of small interfering RNAs siRNAs directed against importin H9252 mRNAs which down regulated classical nuclear import pathways The siRNAs did indeed prime cells for enhanced MHV infection Our findings indicated that protein 6 mediated nuclear import blocks augmented MHV infections but is clearly not the only way that this accessory protein operates to create a milieu conducive to robust virus growth Thus the SARS CoV protein 6 accelerates MHV infections by more than one mechanism Severe acute respiratory syndrome coronavirus SARS CoV is a zoonotic virus that is endemic in bats 33 34 This virus can infect exotic animals marketed for consumption in southeast China 22 58 and in doing so can acquire proximity to human populations SARS CoV entered human hosts in 2002 to 2003 and disseminated rapidly to more than 30 coun tries across five continents killing H11011800 individuals The SARS CoV virion contains a H1101130 kb plus strand RNA ge nome enclosed within a pleiomorphic membrane envelope Many of the features of this virus are known from over 25 years of research on animal coronaviruses The 5H11032H110112 3 of the ge nome encodes two very large polyproteins which are rapidly proteolyzed into components functioning in viral RNA synthe sis and metabolism while the 3H11032H110111 3 includes several virion assembly subunits notably the proteins spike S envelope E membrane M and nucleocapsid N Integrated both between and within these genes encoding virion proteins are eight so called accessory genes 51 These accessory genes originally designated numerically as open reading frames ORFs 3a through 9b were identified in the original 2003 SARS CoV isolate strain Urbani and are now known to be ubiquitous in SARS CoVs obtained from infected bats civit cats raccoon dogs and humans 33 46 64 suggesting evolu tionary conservation of important viral functions The majority of the accessory genes are expressed in SARS CoV infected cells 8 10 28 37 39 48 54 70 Many are membrane pro teins and as antibodies have become available several of the accessory proteins have been detected as copurifying with viri ons 26 48 50 and have been structurally resolved by X ray crystallography 39 Although these studies have provided important informa tion about the SARS CoV accessory proteins their central functions remain largely unknown Indeed accessory protein functions may be difficult to discern as elimination of most of the accessory genes through reverse genetics generates recom binant SARS CoVs with tissue culture growth properties re markably similar to native SARS viruses 1 16 68 Experi mental rodent models for SARS CoV infection and disease have been recently developed 35 45 56 61 and under these experimental conditions recombinant SARS CoVs lacking a subset of accessory genes were nearly equivalent to complete SARS CoV in virulence and pathogenicity 2 14 16 68 Thus at present there is no satisfying explanation of the roles that these evolutionarily conserved accessory proteins play in nat ural animal or human SARS CoV infections Accessory protein 6 has received significant scrutiny as this protein does exhibit functions that may relate to its presumed roles in SARS CoV infections When expressed alone from plasmid cDNA protein 6 ablates type I interferon signaling indicating its potential in thwarting innate immune effectors 15 31 Notably several different coronavirus products may have similar immune evasion activities 31 a redundancy that presumably accounts for the inapparent in vivo consequences of ORF6 elimination from SARS coronavirus However when evaluated in the context of heterologous murine coronavirus infections protein 6 has clearly recognized activities Engi neered recombinant murine coronaviruses constructed to ex press SARS CoV ORF6 designated rJ 2 2 6 recombinant Corresponding author Mailing address Department of Microbi ology and Immunology Loyola University Medical Center 2160 South First Avenue Maywood IL 60153 Phone 708 216 4850 Fax 708 216 9574 E mail tgallag lumc edu H17188 Published ahead of print on 30 April 2008 7212 on March 10 2015 by guest http jvi asm org Downloaded from JHM strain 2 2 V 1 encoding SARS p6 41 have been com pared with isogenic rJ 2 2 6 KO viruses in which ORF6 is closed via knockout KO of the initiation codon Relative to rJ 2 2 6 KO the rJ 2 2 6 viruses grow more rapidly both in tissue culture 55 and in the murine central nervous system and have increased in vivo lethality 40 41 These properties of protein 6 may relate to direct effects on the coronavirus replication machinery as protein 6 colocalizes intracytoplas mically with membrane proximal sites of viral RNA dependent RNA synthesis and does indeed provide for earlier viral RNA synthesis in tissue culture contexts 55 Protein 6 is a 63 amino acid peptide that comprises a rela tively hydrophobic N terminal portion of H1101140 residues and a C terminal hydrophilic extension The N terminal region may function as a signal anchor for membrane association as pro tein 6 has the biochemical characteristics of an integral mem brane protein 55 This membrane associating property of the N terminal region is presumed to operate in assisting develop ment of viral RNA replication sites which are well known to reside on the interfaces between intracellular membranes and cytosol The C terminal region extends from membranes into cytosolic environments 40 and displays several motifs that are vital for a separate and novel activity 15 C terminal residues of protein 6 interact with cellular importins mediators of pro tein import into the nucleus 15 Accumulating protein 6 on cytoplasmic membranes titrates importins to cytoplasmic sites and thereby thwarts cellular capacity to transport cytoplasmic cargo into the nucleus Protein 6 has been shown to impede nuclear import of proteins such as IRF3 and STAT1 key regulators of interferon gene transcription 31 While it is reasonably assumed that this activity of the protein 6 C termi nus contributes to virus growth or pathogenesis by thwarting innate immune responses there are few supporting data In fact alanine substitutions engineered into the C terminus did not reduce the murine neurovirulence of rJ 2 2 6 40 leaving it unclear whether the hydrophilic cytoplasmic tail of protein 6 makes significant contributions to the growth or virulence of this related coronavirus These indications that unique biological activities might be separately ascribed to the N and C terminal regions of protein 6 led us into investigations dissecting the relative contributions of each region to recombinant JHM virus growth In extending a recently published report 15 we found that protein 6 blocked the nuclear accumulation of proteins relying on classical nuclear import pathways with the C terminus being vital in blocking nuclear import More importantly we correlated this nuclear import blockade with enhanced murine hepatitis virus MHV infections giving credence to the hypothesis that this conserved 63 amino acid accessory protein creates superior environments for virus growth by enriching the cytosol with proteins oth erwise destined to the nucleus We found however that this impediment to protein import into the nucleus made only a minor contribution to virus growth as C terminally trun cated forms of protein 6 lacking this activity still retained the majority of virus augmenting function These findings indicate that this small protein 6 supports murine corona virus infections by more than one mechanism MATERIALS AND METHODS Cells Human epithelial kidney cells HEK293 baby hamster kidney cells BHK and BHK cells stably expressing T7 polymerase gene BSR T7 5 were grown and maintained in Dulbecco modified Eagle medium DMEM supple mented with 10 fetal bovine serum FBS 10 mM Na HEPES 2 mM L glutamine and penicillin 100 U ml streptomycin 100 H9262g ml Mouse fibroblast cells 17Cl 1 were grown and maintained in DMEM supplemented with 5 tryptose phosphate broth 5 FBS and penicillin streptomycin Plasmid constructions Expression plasmids encoding full length protein 6 pCAGGS 6 and pBMN 6 pBMN 6 IRES eGFP and C terminal deletion mu tant pBMN 6H9004C pBMN 6H9004C IRES eGFP lacking C terminal residues 53 to 63 were constructed using standard PCR techniques The retroviral vector pBMN IRES eGFP was kindly provided by Garry P Nolan Stanford University Stanford CA 30 All clones carry a hemagglutinin HA tag sequence YPY DVPDYA at C termini for easy detection of expressed protein by Western blot and immunofluorescence assays Renilla luciferase reporter plasmid pRL TK which contains Renilla luciferase cDNA under the herpes simplex virus thymi dine kinase TK promoter was purchased from Promega Madison WI and firefly luciferase reporter plasmid pECMVT7Luc containing the firefly lucifer ase gene under control of a bacteriophage T7 promoter has been described previously 3 The plasmids encoding the enhanced green fluorescent protein EGFP trimer with or without the nuclear localization signal NLS were created using methods developed by Genove et al with some modifications 18 Briefly an EGFP cassette was PCR amplified using forward primer 5H11032 ATCTCGAGTGAGCAA GGGCGAGGAGC 3H11032 and reverse primer 5H11032 GAGAATTCAGCAAGGGCGA GGAGCTG 3H11032 and ligated into pEGFP C1 Clontech Inc to generate a con struct we designate p2xEGFP A third EGFP cassette was PCR amplified using forward primer 5H11032 CTGAATTCTCCGGACTTGTACAGCAGG 3H11032 and reverse primer 5H11032 TTGTCGACGTCCGGACTTGTACAGCTCG 3H11032 and cloned into p2xEGFP to yield p3xEGFP Subsequently oligonucleotides encoding two tan dem copies of the simian virus 40 SV40 large T antigen NLS and human heterogeneous ribonucleoprotein hnRNPA1 nuclear localization signal M9 NLS were subcloned at the 3H11032 end of the third GFP open reading frame to yield p3xEGFP SV40 NLS and p3xEGFP M9 NLS respectively The p3xEGFP SV40 NLS encodes an H1101184 kDa fluorescent protein with a C terminal appended SV40 NLS ELYKSGRRAQDPKKKRKVDPKKKRKV the C terminal GFP residues are shown in bold linker residues are in italics and the NLS is underlined The p3xEGFP M9 NLS encodes a fluorescent protein with C terminal appended M9 NLS ELYKSGRRAQGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYF AKPRNQGGY with C terminal GFP residues in bold linker residues in italics and the NLS underlined Transient transfection and infection 293T cells were grown in 10 cm 2 dishes and transiently transfected with 2 0 H9262g pcDNA mCEACAM1a MHV receptor J Lacny and T M Gallagher unpublished data and 1 H9262g of p6 encoding plasmid pCAGGS 6 pCAGGS 6 UT untagged pBMN 6 pBMN 6H9004C or vec tor control using a calcium phosphate transfection method 47 After 24 h transfection media were removed and cells were infected with recombinant MHV strain JHM rJ 2 2 or rJ2 2 expressing SARS CoV protein 6 rJ2 2 6 at a multiplicity of infection of 0 2 PFU cell in serum free DMEM After a 1 h adsorption period the inocula were removed and infections were allowed to proceed overnight in DMEM 10 FBS Plaque assay and immunoblotting of virion proteins Culture media were collected at 16 h postinfection or at other times postinfection as indicated and clarified at 2 000 H11003g for 20 min In plaque assays aliquots of clarified media were serially diluted and applied to monolayers of 17Cl 1 cells After1hofadsorption inocula were removed and cells were washed with phosphate buffered saline PBS and then overlaid with DMEM containing 1 FBS and 0 5 agar Becton Dickinson MD The cells were fixed at 3 days postinfection and stained with 1 crystal violet and plaques were enumerated For isolation and detection of virus particles clarified medium 0 7 ml was overlaid on 0 6 ml cushions of 20 sucrose 25 mM Na HEPES pH 7 4 100 mM NaCl 0 01 bovine serum albu min Virions were pelleted by ultracentrifugation at 200 000 H11003 g for 30 min Virion containing pellets were dissolved in sodium dodecyl sulfate SDS solu bilizer 50 mM Tris HCl pH 6 8 10 glycerol 2 SDS 5 2 mercaptoethanol and 0 1 bromophenol blue fractionated using SDS polyacrylamide gel elec trophoresis PAGE and immunoblotted with antibodies reacting with the HA epitope Covance Inc Berkeley CA S proteins monoclonal antibody MAb 10G provided by Fumihiro Taguchi M proteins MAb J 1 3 and N proteins MAb J 3 1 provided by John O Fleming Transient transfection and luciferase assay To evaluate the influence of protein 6 on reporter gene expression BHK 293T and BSR T7 cells were VOL 82 2008 MECHANISMS ENHANCING MHV INFECTION 7213 on March 10 2015 by guest http jvi asm org Downloaded from cotransfected with 1 H9262g of reporter plasmid pRL TK or pECMVT7Luc or both and 10 fold increasing concentrations of pCAGGS 6 The total amount of DNA was kept the same for each transfection mixture by adding empty pCAGGS MCS vector In 293T cells transfections were performed with calcium phosphate transfection reagents as described elsewhere 9 47 whereas in BHK and BSR T7 cells transfections were performed with Lipofectamine 2000 as recom mended by the manufacturer Invitrogen Corporation At 24 h posttransfection hpt cells were rinsed twice in PBS and lysed in passive reporter lysis buffer Promega after resuspension at 5 H11003 10 6 cells per ml Five H9262l aliquots were assayed using luciferase assay reagent Promega and luminescence was re corded using a Veritas Microplate luminometer Turner Biosystems Sunnyvale CA All transfections were performed in triplicate samples were measured in quadruplicate and numerical uncertainties are shown by error bars Immunofluorescence and confocal microscopy To assess the nucleo cytoplas mic distribution of NLS containing GFP 293T cells were grown on glass cover slips placed into 10 cm 2 dishes and transfected with plasmids encoding reporter genes p3xGFP M9 NLS p3xGFP SV40 NLS or p3xGFP along with p6 expres sion plasmids pCAGGS 6 pBMN 6 and pBMN 6H9004C or vector control plas mids Cells were fixed at 24 hpt in 4 paraformaldehyde for 10 min and per meabilized in digitonin buffer 300 mM sucrose 100 mM KCl 2 5 mM MgCl 2 1 mM EDTA 10 mM HEPES pH 6 9 containing 5 H9262g ml digitonin for 15 min at room temperature The coverslips were incubated in blocking buffer 2 bovine serum albumin in PBS for 15 min followed by an overnight incubation with primary antibody directed against HA tag at a dilution of 1 5 000 The coverslips were washed three times in ice cold PBS at room temperature and incubated with fluorochrome conjugated secondary antibodies all from Molecular Probes Inc Eugene OR for1hatroom temperature The coverslips were washed three times in PBS and mounted with ProLong Gold antifade reagent Invitro gen Corporation Confocal images were captured using a Carl Zeiss model 510 laser scanning confocal microscope Digitized images were processed with Image J National Institutes of Health siRNA transfection and MHV infections Predesigned small interfering RNAs siRNAs catalog number AM16704 siRNA ID 11218 11125 and 145041 cor responding to exons 3 and 9 of human importin H9252 mRNA accession number NM 002265 negative control siRNA catalog number 4611 and scrambled control siRNA GGCACAAUAUCAGCAGCGG catalog number AM16104 were purchased from Ambion Austin TX These siRNAs were transfected into 239T cells in graded doses as indicated along with pcDNA mCEACAM1a using Lipofectamine 2000 Invitrogen Typical experiments involved transfect ing H110112 H11003 10 5 293T cells with 100 nmol of siRNA and 1 5 H9262g of pcDNA mCEACAM1a in 1 ml of OPTI MEM Life Technologies Controls included mock transfection transfection reagent lacking siRNA negative control siRNA and scrambled control siRNA At 2 days posttransfection cells were infected with rJ 2 2 After 16 h supernatants were collected and infectivities were evaluated by plaque assay Transfections were done in triplicate and each ex periment was repeated two to three times RESULTS Plasmid DNAs encoding protein 6 prime cells for acceler ated MHV infections In our previous studies we used recom binant MHVs with SARS CoV open reading frames integrated in the genome Using these chimeric recombinant viruses we showed that the SARS CoV protein 6 was a virulence factor increasing both MHV yields in tissue culture and neuroviru lence in mice 41 55 In the present study we employed a related but distinct approach Here cultured cells were tran siently transfected with expression plasmids encoding wild type and mutant protein 6 and then subsequently infected with different MHV strains This approach was simpler than our previous approach because it did not require development of recombinant viruses and was also able to address whether protein 6 might independently modify the cell in ways that influence the subsequent infection process 293 cells were transfected with the desired plasmids and subsequently in fected with recombinant MHV rJ2 2 or recombinant MHV expressing SARS CoV protein 6 rJ2 2 6 Relative to vector transfected cells we observed robust viral egress from p6 expressing cells as registered by abundant Western blot signals corresponding to virion proteins S N and M Fig 1A These secreted particles were infectious as demonstrated by plaque assay results revealing H1101110 fold more infectivity in medium from p6 expressing cells relative to vector controls Fig 1B Comparative analysis of HA tagged and untagged protein 6 revealed that the HA epitope had no influence on p6 activity Fig 1A and B This augmenting effect of p6 was evident as early as 8 to 10 h postinfection Fig 1C and D In a parallel experiment 293 cells were transfected with plasmids encoding the SARS accessory proteins 3a 3b and 7a 24 26 29 50 69 71 and then infected with MHV Immu noblot analyses confirmed that the plasmids were expressed at levels comparable to protein 6 however none had any signif icant impact on MHV infection kinetics Fig 1A and B and data not shown These data unequivocally indicated that pro tein 6 creates cellular environments that benefit MHV growth however the underlying mechanism responsible for this accel erated infection remained unclear Protein 6 reduces expression from plasmids requiring tran scription in the nucleus Clues to the mechanism by which protein 6 increases MHV growth came from the u