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JOURNAL OF VIROLOGY Nov 2005 p 13285 13297 Vol 79 No 21 0022 538X 05 08 00H110010 doi 10 1128 JVI 79 21 13285 13297 2005 Copyright 2005 American Society for Microbiology All Rights Reserved A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein Kelley R Hurst Lili Kuo Cheri A Koetzner Rong Ye Bilan Hsue and Paul S Masters Wadsworth Center New York State Department of Health Albany New York 12201 Received 6 June 2005 Accepted 3 August 2005 The two major constituents of coronavirus virions are the membrane M and nucleocapsid N proteins The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino terminal ectodomain and a large carboxy terminal endodomain The M endodomain interacts with the viral nucleocapsid which consists of the positive strand RNA genome helically encapsidated by N protein monomers In previous work with the coronavirus mouse hepatitis virus MHV a highly defective M protein mutant MH90042 was constructed This mutant contained a 2 amino acid carboxy terminal truncation of the M protein Analysis of second site revertants of MH90042 revealed mutations in the carboxy terminal region of the N protein that compensated for the defect in the M protein To seek further genetic evidence corroborating this interaction we generated a comprehensive set of clustered charged to alanine mutants in the carboxy terminal domain 3 of N protein One of these mutants CCA4 had a highly defective phenotype similar to that of MH90042 Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss of function mutation rather than a dominant negative mutation Analysis of multiple second site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N These data more precisely define the region of the N protein that interacts with the M protein Further we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions Coronavirus assembly results from an accumulation of in teractions among four structural proteins the positive sense RNA genome and a host membrane envelope obtained from the site of budding which is the endoplasmic reticulum Golgi intermediate compartment Three of the four structural pro teins are embedded in the virion envelope the spike protein S the membrane protein M and the small envelope pro tein E The fourth the nucleocapsid protein N resides in the virion interior wrapping the positive strand RNA genome into a helical nucleocapsid The most abundant viral constituent is M a 25 kDa protein containing three transmembrane segments flanked by a short amino terminal ectodomain and a large carboxy terminal endodomain The M protein is the central organizer of assem bly in that it self associates 8 10 21 captures S for virion incorporation 36 37 and selectively packages the fraction of N protein that is bound to genomic RNA 32 34 The S protein is responsible for viral attachment to host cell recep tors and for the membrane fusion event that initiates infection This type I membrane protein shortly after its synthesis fold ing and oligomerization forms complexes with M protein in the endoplasmic reticulum 36 37 The M interacting domain of the S protein was localized to the transmembrane endodo main region of the molecule based on the assembly of chi meric S proteins into virus like particles VLPs 16 or virions 18 23 More recently this determinant was further localized to the endodomain of S which could confer virion assembly competence if transferred to a heterologous transmembrane protein 48 The general 1 3 4 31 45 but not universal 20 consen sus from studies of VLPs is that formation of coronaviruses is mediated by just the M and the E proteins and that neither S protein nor the nucleocapsid plays an obligate role in virion morphogenesis The precise role of E protein in this process is enigmatic with some evidence pointing to a direct interaction between E and M 1 5 and other observations suggesting that E acts independently of M in the budding compartment 4 26 41 A very recent finding that may shed light on the workings of the E protein is the demonstration that the E protein of severe acute respiratory syndrome coronavirus has the prop erties of a cation selective ion channel 47 The construction of E protein mutants of mouse hepatitis virus MHV has confirmed the critical role of E in viral assembly 14 Surpris ingly however MHV remains viable although severely im paired following deletion of the E gene 25 whereas disrup tion of the E gene of porcine transmissible gastroenteritis virus TGEV is lethal 6 38 The interaction between M protein and N protein has been previously explored by biochemical and molecular biological methods for both MHV 33 34 44 and TGEV 12 In a genetic approach we constructed and analyzed a highly defec tive MHV mutant with a carboxy terminal truncated M pro tein MH90042 and we identified suppressors of this mutation that Corresponding author Mailing address David Axelrod Institute Wadsworth Center NYSDOH New Scotland Avenue P O Box 22002 Albany NY 12201 2002 Phone 518 474 1283 Fax 518 473 1326 E mail masters wadsworth org Present address Stratagene 11011 N Torrey Pines Rd La Jolla CA 92037 13285 on May 8 2015 by North Carolina State University Libraries http jvi asm org Downloaded from mapped to the carboxy terminus of the N protein 24 This led us to more closely examine which amino acids in N protein determine its recognition by M protein Based on interstrain sequence comparisons the MHV N protein has been proposed to comprise three conserved domains separated by two highly divergent spacer regions A and B 39 Domains 1 and 2 which make up most of the molecule are very basic with the RNA binding property of N mapping to domain 2 27 35 In contrast domain 3 the carboxy terminal 45 amino acids of N has an excess of acidic residues In the present study we car ried out a systematic mutagenesis of domain 3 and we iden tified an adjacent pair of functionally essential aspartates anal ysis of which revealed further genetic cross talk between the carboxy termini of the N and M proteins In addition we found that fusion of N protein domain 3 to the carboxy terminus of a heterologous protein caused it to be selectively incorporated into MHV virions MATERIALS AND METHODS Cells and viruses Wild type MHV strain A59 and mutants were propagated in mouse 17 clone 1 17Cl1 or L2 cells plaque assays and plaque purifications of mutant recombinants and revertants were carried out in mouse L2 cells The interspecies chimeric virus fMHV v2 17 was grown in feline FCWF cells or AK D fetal lung cells Plasmid constructs Clustered charged to alanine CCA mutants 1 through 6 were originally constructed in pB36 29 which is a donor RNA transcription vector that encodes an RNA comprising the 5H11032 end of the MHV genome 467 nucleotides nt fused via a 49 nt linker to the 3H11032 end of the genome beginning at the start codon of the N gene Mutations were generated by splicing overlap extension PCR 19 and were inserted into pB36 by exchange of the segment between the AccI site in spacer B and the BstEII site in the 3H11032 untranslated region 3H11032 UTR 29 producing vectors pBL56 through pBL61 respectively Of note for subsequently obtained results the CCA4 mutations in pBL59 changed N gene codons 440 and 441 from GAU D to GCU A In CCA1 plasmid pBL56 an additional silent mutation was inadvertently generated changing N gene codon 447 from GGG to GGA All other mutants were constructed in pMH54 23 or pSG6 17 which are donor RNA transcription vectors that encode RNAs comprising the 5H11032 end of the MHV genome 467 nt fused via a 73 nt linker to the 3H11032 end of the genome beginning at codon 28 of the HE pseudogene Plasmid pSG6 is identical to pMH54 except for a coding silent BspEI site that spans codons 444 to 446 of the N gene 17 The CCA4 mutations of pBL59 were transferred to pMH54 by exchange of the segment between the NheI site in domain 2 of the N gene and the BclI site in the 3H11032 UTR to produce vector pLK94 For the purpose of reconstructing intragenic CCA4 revertants a reverse transcription PCR RT PCR product generated from revertant 2 and containing the I438S D440A and D441A mutations was cloned between the NheI and BclI sites of pSG6 to produce vector pKRH5 Similarly an RT PCR product generated from revertant 17 and containing the Q437L I438S D440A and D441A mutations was cloned between the NheI and BclI sites of pSG6 For the purpose of reconstructing intergenic CCA4 revertants individual RT PCR products generated from cho sen revertants were incorporated into pKRH5 via unique restriction sites For the M gene mutations I128T revertant 1 Y143H revertant 9 and Y156H revertant 5 an RT PCR product was cloned between the EagI and BssHII sites occurring in the M gene ectodomain and endodomain respectively For the M gene mutation V202I revertant 7 an RT PCR product was cloned between the M gene BssHII and N gene NheI sites Transcription vector pA112 containing a duplication of the N gene was constructed as a precursor to the previously described pA122 11 which con tains a single copy of the N gene transposed to the position occupied by gene 4 in the wild type MHV genome The sequences of the upstream and downstream boundaries of the transposed N gene are given as junctions 5 and 6 in Fig 3 of reference 11 In the upstream copy of the N gene in pA112 the hemagglutinin HA epitope tag YPYDVPDYA replaces amino acids 386 to 394 of spacer B and a His 6 tag is appended to the carboxy terminus Details of the construction of pA112 and deletion derivatives will be reported elsewhere C A Koetzner and P S Masters unpublished results CCA4 mutations in the duplicated copy of the N gene were created in a subclone by PCR mutagenesis with simultaneous elimination of the His 6 tag and these were shuttled into pA112 via unique Sse8387I and XbaI sites flanking the upstream copy of the N gene Donor RNA transcription vectors encoding derivatives of the gene for green fluorescent protein GFP replacing most of gene 4 of MHV were constructed from pMH54GFP 7 generously provided by Jayasri Das Sarma and Susan Weiss University of Pennsylvania School of Medicine N protein domain 3 in construct pMH54GFP d3 or N protein spacer B plus domain 3 in construct pMH54GFP Bd3 was fused to the GFP open reading frame ORF by three way ligations making use of the unique SalI and NotI sites bounding the GFP gene in pMH54GFP 7 as well as a BsrGI site near the carboxy terminus of the GFP ORF In each case the BsrGI NotI fragment incorporating the N gene segment was generated by PCR and a two amino acid spacer SG creating a unique BspEI site was added between the GFP ORF and the start of the N gene segment Derivatives of pMH54GFP Bd3 containing the CCA4 mutations were then made by PCR mutagenesis replacing the BspEI NotI fragment encompass ing the N gene segment To bacterially produce maltose binding protein fused to the carboxy terminus of the N protein the region of the N gene encoding spacer B plus domain 3 was cloned between the BamHI and HindIII sites of vector pMAL p2 New England Biolabs The fusion protein designated MBP Bd3 and control maltose binding protein were inducibly expressed in Rosetta DE3 pLysS cells Novagen All manipulations of DNA were carried out by standard methods 42 The compositions of all plasmid constructs were initially verified by restriction anal ysis Then all cloned cDNA precursors PCR generated segments and newly reconstructed or created junctions of each plasmid were confirmed by automated DNA sequencing Mutant construction by targeted RNA recombination Mutants CCA1 CCA2 CCA3 CCA5 and CCA6 were created by targeted recombination between donor RNAs from pB36 derived transcription vectors and the recipient virus Alb4 22 as described previously 29 All other viral mutants were obtained from targeted recombination using the interspecies chimera fMHV v2 as the recipient virus as described in detail previously 17 23 25 In brief monolayers of feline AK D or FCWF cells were infected with fMHV v2 and were subse quently transfected with capped synthetic donor RNA by electroporation Gene PulserII Bio Rad Donor RNAs were generated by transcription with T7 RNA polymerase mMessage mMachine Ambion using PacI truncated plasmid tem plates The infected and transfected feline cells either were overlaid onto murine cell monolayers or else were directly plated into 10 cm 2 wells Released progeny virus was harvested at 24 to 48 h postinfection at 37 C Recombinant candidates were selected and purified by two rounds of plaque titration on murine L2 cell monolayers at 37 C For analysis of each recombinant candidate total RNA was extracted from infected 17Cl1 cell monolayers Ultraspec reagent Biotecx and reverse tran scription of RNA was carried out with a random hexanucleotide primer and avian myeloblastosis virus reverse transcriptase Life Sciences To ascertain the presence of incorporated mutations or genes PCR amplifications of cDNAs were performed with AmpliTaq polymerase Roche using primer pairs flanking the relevant regions of the genome RT PCR products were analyzed directly by agarose gel electrophoresis and were purified with Quantum prep columns Bio Rad prior to automated sequencing Western blotting For preparation of lysates confluent monolayers 10 to 25 cm 2 of 17Cl1 cells either were mock infected or were infected with wild type MHV or constructed mutants cells were then incubated at 33 C or 37 C At 6 to 18 h postinfection monolayers were washed twice with phosphate buffered saline and then lysed by addition of 600 H9262l of 50 mM Tris HCl pH 8 0 150 mM NaCl 1 0 Nonidet P 40 0 7 H9262g ml pepstatin 1 0 H9262g ml leupeptin 1 0 H9262g ml aprotinin and 0 5 mg ml Pefabloc SC Roche Lysates were held for 5 to 15 min on ice and were then clarified by centrifugation For analysis of proteins assembled into MHV virions viruses were purified by polyethylene glycol precipitation followed by two cycles of equilibrium centrifugation on potassium tartrate glycerol gradi ents as described in detail previously 48 Samples of either infected cell lysates or purified virions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 10 or 12 polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane Blots were probed with one of the fol lowing antibodies anti N monoclonal antibody MAb J 3 3 anti M MAb J 1 3 MAbs J 3 3 and J 1 3 were generously provided by John Fleming University of Wisconsin Madison an anti HA epitope tag MAb MAb 12CA5 Roche a polyclonal rabbit antiserum raised against a bacterially expressed maltose bind ing protein MHV N protein fusion or an anti GFP MAb BD Biosciences Bound primary antibodies were visualized using a chemiluminescent detection system ECL Amersham 13286 HURST ET AL J VIROL on May 8 2015 by North Carolina State University Libraries http jvi asm org Downloaded from RESULTS Isolation of mutants in domain 3 of the N protein In pre vious work our laboratory constructed a highly defective M protein mutant MH90042 in which the carboxy terminal two amino acids of M protein were truncated 24 Analysis of second site revertants of MH90042 demonstrated that certain mutations in the carboxy terminal region of the N protein could compensate for the M protein truncation suggesting a direct interaction be tween these two protein domains To further examine the function of domain 3 of the MHV N protein we generated a comprehensive set of six CCA mutants spanning domain 3 generally following the algorithm of Wertman and coworkers 46 In each case where two or more charged amino acids occurred within a sliding window of five or six residues a mutant was designed that replaced all charged residues with alanines Fig 1A This mutagenesis strategy tests the assump tion that linear clusters of charged amino acids are found mainly on the surfaces of proteins where they can potentially make strong contributions to protein protein interactions The six CCA mutants were isolated by targeted RNA re combination a method that is used for the site specific intro duction of mutations into coronaviruses via recombination be tween a synthetic donor RNA and a recipient virus that can be counterselected 28 30 CCA mutants 1 2 3 5 and 6 were obtained by an earlier version of targeted recombination that made use of selection against a thermolabile recipient virus Alb4 22 29 These mutants were phenotypically indistin guishable from the wild type with the exception of mutant CCA3 which formed somewhat smaller plaques than the wild type at all temperatures tested data not shown Despite re peated attempts however we could not obtain mutant CCA4 from Alb4 Additional attempts were made to obtain CCA4 following the development of a host range based selective sys tem for targeted RNA recombination This system makes use of an interspecies chimera named fMHV which has the spike ectodomain of feline infectious peritonitis virus FIPV in place of that of MHV As a result fMHV grows in feline cells but not in mouse cells This tropism is reversed by targeted FIG 1 Construction of a comprehensive set of CCA mutants in domain 3 of the N protein of MHV A Schematic of the proposed structure of MHV N protein with three domains separated by two short spacers designated A and B 22 29 39 40 The expanded region of amino acid sequence shows the carboxy terminus of N protein including part of spacer B and all of domain 3 Residue numbers and charged residues are indicated above the wild type sequence for CCA mutants 1 through 6 only those residues that differ from the wild type are shown B Strategy for selection of mutant CCA4 by targeted RNA recombination between the interspecies chimera fMHV v2 17 and donor RNA transcribed from a derivative of plasmid pMH54 23 fMHV v2 contains the ectodomain encoding region of the FIPV S gene shaded rectangle and grows in feline cells but not in murine cells A single crossover within the HE gene should generate a recombinant that has simultaneously reacquired the MHV S ectodomain and the ability to grow in murine cells and has also incorporated the mutations in the N gene star The rearranged order of genes following the S gene in fMHV v2 precludes the occurrence of a secondary crossover event downstream of the S gene 17 C Plaques of purified mutant CCA4 Alb334 compared to the reconstructed wild type Alb240 25 Plaque titrations were carried out on mouse L2 cells at 37 C Monolayers were stained with neutral red at 72 h postinfection and were photographed 18 h later VOL 79 2005 INTERACTION BETWEEN CORONAVIRUS N AND M PROTEINS 13287 on May 8 2015 by North Carolina State University Libraries http jvi asm org Downloaded from recombination with a donor RNA that restores the MHV S ectodomain and simultaneously transduces additional con structed mutations into the MHV genome Resulting mutants are selected on the basis of the restoration of the ability of the virus to grow in mouse cells 23 24 In a recent version of this system the genes downstream of S have been rearranged in the recipient virus fMHV v2 Fig 1B thereby effectively eliminat ing the possibility of downstream crossover events that could exclude the mutation s of interest 17 A total of 12 independent targeted recombination experi ments with donor RNA containing the CCA4 mutations were conducted in three separate sets in which wild type donor RNA yielded robust numbers of control recombinants In only one of these trials were we able to recover three tiny plaque siblings designated Alb334 Alb335 and Alb340 All three of these mutant candidates were confirmed to have the two point mutations creating the residue changes D440A and D441A expected for CCA4 However all three also conta