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JOURNAL OF VIROLOGY 0022 538X 00 04 0010 Aug 2000 p 6911 6921 Vol 74 No 15 Copyright 2000 American Society for Microbiology All Rights Reserved Characterization of an Essential RNA Secondary Structure in the 39 Untranslated Region of the Murine Coronavirus Genome BILAN HSUE 1 2 TOINETTE HARTSHORNE 3 AND PAUL S MASTERS 1 2 Wadsworth Center for Laboratories and Research New York State Department of Health 1 and Department of Biomedical Sciences University at Albany State University of New York 2 Albany New York 12201 and Center for Immunology and Microbial Disease Albany Medical College Albany New York 12208 3 Received 13 March 2000 Accepted 8 May 2000 We have previously identified a functionally essential bulged stem loop in the 3 untranslated region of the positive stranded RNA genome of mouse hepatitis virus This 68 nucleotide structure is composed of six stem segments interrupted by five bulges and its structure but not its primary sequence is entirely conserved in the related bovine coronavirus The functional importance of individual stem segments of this stem loop was characterized by genetic analysis using targeted RNA recombination We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs These studies were complemented by enzymatic and chemical probing of the stem loop Taken together our results confirmed most of the previously proposed structure but they revealed that the terminal loop and an internal loop are larger than originally thought Three of the stem segments were found to be essential for viral replication Further our results suggest that the stem segment at the base of the stem loop is an alternative base pairing structure for part of a downstream and partially overlapping RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication Mouse hepatitis virus MHV one of the best characterized members of the coronavirus family has a single stranded pos itive sense RNA genome some 31 kb in length Upon infection the first two thirds of this exceptional molecule is translated into an RNA dependent RNA polymerase Coronavirus RNA synthesis then proceeds by a unique and incompletely under stood mechanism described by conflicting models 1 16 24 44 46 54 55 60 Initially the genomic RNA becomes the template for at the least a full length negative sense anti genome Further events produce a series of smaller sub genomic RNAs of both polarities The positive sense sub genomic RNAs form a 39 nested set with each containing a 70 nucleotide nt leader that is identical to the 59 end of the genome and is joined at a downstream site to a stretch of sequence identical to the 39 end of the genome The negative sense subgenomic RNAs form a 59 nested set and are roughly 1 10 to 1 100 as abundant as their positive sense counterparts with each possessing the complement of this arrangement including a 59 oligo U tract and a 39 antileader 10 47 Many advances in investigating the mechanism of coronavi rus RNA synthesis have been enabled by the discovery of defective interfering DI RNAs of MHV 29 30 52 and of other coronaviruses 5 34 39 DI RNAs are extensively de leted genomic remnants that replicate by using the RNA syn thesis machinery of a helper virus often interfering with viral genomic RNA replication Studies of naturally occurring and artificially constructed DI RNAs which can be transfected into helper virus infected cells have mapped cis acting sequence elements from the genome that participate in replication and transcription These deletion analyses have demonstrated that the minimal extent of the 39 terminus of the MHV genome that is able to sustain DI RNA replication falls between 417 and 463 nt 17 25 28 53 Notably this includes a portion of the upstream nucleocapsid N gene as well as the entire 301 nt 39 untranslated region 39 UTR Given this requirement it was surprising when further study showed that the minimal tract of template required for negative strand RNA synthesis is con tained within just the last 55 nt at the 39 end of the genome in addition to an as yet undetermined amount of poly A tail 26 This suggested that sequences upstream of the negative strand promoter are required for positive strand RNA synthe sis the initiation of which may thus require a circularizing interaction between the 59 and 39 termini of the viral genome 26 Although a full length infectious cDNA clone of MHV has not yet been attained some avenues into coronavirus genetics have been made possible through the development of site directed mutagenesis by targeted RNA recombination 20 22 31 40 42 This technique allows the incorporation of muta tions into the coronavirus genome via RNA RNA recombina tion between a synthetic donor RNA and the genome of a recipient virus that can be selected against Recently in at tempting to replace the MHV 39 UTR with its counterpart from the bovine coronavirus BCoV genome by targeted RNA recombination we found that the two 39 UTRs were fully interchangeable 12 Moreover we discovered that a pre dicted bulged stem loop secondary structure adjacent to the stop codon of the N gene is essential for viral replication Fig 1 12 This 68 nt structure is composed of six stem segments interrupted by five bulges which range from 1 to 4 nt Se quence comparison suggests that 8 of the 10 nt that are differ ent between MHV and BCoV in this region form 4 covariant bp falling in two of the stem segments Our previous molecular genetic analysis suggested that the base pairing but not the primary sequence of these 4 covariant bp is necessary for replication 12 In this report we determined the functional significance of each of the putative stem segments and we probed the RNA secondary structure of this region by chem ical modification and enzymatic analysis Our results point to a modification of the originally proposed stem loop and they suggest a relationship between this structure and an immedi Corresponding author Mailing address David Axelrod Institute Wadsworth Center NYSDOH New Scotland Ave P O Box 22002 Albany NY 12201 2002 Phone 518 474 1283 Fax 518 473 1326 E mail masters wadsworth org 6911 on April 13 2015 by UNIV OF CONNECTICUT http jvi asm org Downloaded from ately downstream pseudoknot that has been recently described for the 39 UTR of BCoV 56 MATERIALS AND METHODS Virus and cells Growth of all stocks of MHV A59 wild type mutant and recombinant viruses was carried out in mouse 17 clone 1 17Cl1 cells All plaque titrations and plaque purifications were performed with mouse L2 cells Spinner cultures of L2 cells were maintained for RNA transfection by electroporation as described previously 32 The interspecies chimeric coronavirus fMHV was grown in feline FCWF cells 22 Plasmid constructs To enable more rapid construction of mutations in the 39 UTR convenient restriction sites were incorporated into plasmid pB36 a T7 transcription vector that encodes a replicating MHV DI RNA comprising the 59 467 nt of the MHV genome followed by a heterologous 48 nt linker the entire N gene the 39 UTR and a 115 residue poly A tail 32 A unique MluI site was created 9 through 14 nt upstream of the N gene stop codon and a unique EcoRV site was created 10 through 15 nt downstream of the bulged stem loop structure see Fig 2A The MluI site was generated by PCR with a mutagenic primer coupled with a primer flanking the upstream AccI site A second DNA fragment containing both the engineered MluI and EcoRV sites and the downstream SacI site was generated in two steps by splicing overlap extension PCR 11 The former PCR product was digested with AccI and MluI the latter was digested with MluI and SacI and the two resulting fragments were inserted via a three way ligation in place of the corresponding AccI SacI region of pB36 to yield pBL85 Fig 2A A series of 15 vectors containing mutations in the bulged stem loop between the MluI and EcoRV sites Fig 1 and 3A were then constructed by cassette mutagenesis For left arm mutants MBL MDL and MFL a pair of mutation containing oligonucleotides bounded by MluI and BstEII sites was ligated with BsiWI MluI and BstEII BsiWI fragments from pBL85 For right arm mutants MAR MBR MCR MDR MER and MFR a pair of mutation containing oligonucleotides bounded by BstEII and EcoRV sites was ligated with SpeI BstEII and EcoRV SpeI fragments from pBL85 For double arm mutants MALR MBLR MCLR MDLR MELR and MFLR the SpeI BstEII fragment from a left arm mutant or the MluI BstEII oligonucleotide cassette and the BstEII HindIII fragment from its right arm mutant counterpart were swapped with the corresponding region of pBL85 via a three way ligation Another set of bulged stem loop mutants were constructed in which the sequence downstream of the EcoRV site was derived from the BCoV 39 UTR rather than the MHV 39 UTR This segment of the BCoV 39 UTR flanked upstream by a primer generated EcoRV site and downstream by an MscI site was produced through PCR amplification of template pBL34 which contains this region of the BCoV 39 UTR 12 The EcoRV MscI PCR fragment was inserted in place of the corresponding region of MCR MFR and MFLR to generate BCR BFR and BFLR respectively For the remaining constructs the EcoRV HindIII fragment of BFR was used to replace the same region in MDR MDL MER MELR and MFL to generate BDR BDL BER BELR and BFL respectively For the subset of the original 15 bulged stem loop mutants that did not give rise to recombinant viruses the same mutations were introduced into another vector pMH54 22 pMH54 is a T7 transcription vector containing the 59 467 nt of the MHV genome connected by a heterologous 72 nt linker to the 39 8 6 kb of the MHV genome beginning at codon 28 of the hemagglutinin HE gene see Fig 2C 22 For mutants MCR MDR MDL MFR MFL and MFLR the NheI SacI fragment of the corresponding pBL85 derived construct was trans ferred to the larger vector through a three way ligation with the SacII NheI and SacI SacII fragments from pMH54 DNA manipulations were carried out by standard methods 41 The se quences of all junctions created by ligations and all segments generated by PCR were verified by dideoxy sequencing 43 using a modified T7 DNA polymerase kit Sequenase Amersham Targeted recombination Donor RNAs were transcribed from pBL85 derived plasmids truncated with HindIII Mutations in these shorter synthetic donor RNAs were incorporated into the MHV genome by targeted recombination with the recipient virus Alb4 exactly as described previously see Fig 2B 12 20 32 Negative results that were obtained with a subset of mutant donor RNAs by this method were then confirmed by attempting targeted recombination with donor RNAs transcribed from PacI truncated vectors derived from pMH54 In this case the interspecies chimeric coronavirus fMHV was used as the recipient virus see Fig 2C 22 Briefly confluent feline FCWF cells were infected with fMHV at a multiplicity of approximately 1 PFU per cell for5hat37 C Infected monolayers were then suspended by trypsin treatment washed in calcium and magnesium free phosphate buffered saline and transfected with donor RNA by electroporation with two consecutive pulses at 960 mF and 0 3 kV using a Bio Rad Gene Pulser Infected and transfected cells were then plated onto monolayers of murine 17Cl1 cells After 48 h of incubation at 37 C progeny viruses were harvested and candidate recombinants were analyzed following two rounds of plaque purification on L2 cells Cytoplasmic RNA from infected 17Cl1 cell monolayers was purified either by a Nonidet P 40 gentle lysis method 18 or with Ultraspec reagent Biotecx per the manufacturer s instructions Direct RNA sequencing was performed by a modification of a dideoxy chain termination procedure using avian myeloblas tosis virus reverse transcriptase Life Sciences 6 38 Radiolabeling of viral RNA and analysis of DI RNA replication Metabolic labeling of virus specific RNA was carried out as previously described 12 32 In brief L2 cells in spinner culture were infected with wild type MHV at a multi plicity of 1 PFU per cell At 2 h postinfection DI RNA was introduced into cells by electroporation and cells were then plated onto a 20 cm 2 monolayer of 17Cl1 cells which was incubated at 37 C until the monolayer developed approximately 50 syncytia Cells were starved for2hinEagle s minimal essential medium containing 5 dialyzed fetal bovine serum and 1 10 of the normal phosphate concentration Cells were then labeled for 2 h with 33 P orthophosphate in phosphate free Eagle s minimal essential medium containing 5 dialyzed fetal bovine serum and 20 mg of actinomycin D Sigma per ml Purified cytoplasmic RNA samples containing equal amounts of radioactivity were analyzed by elec trophoresis on 1 agarose gels containing formaldehyde To analyze DI RNA specific negative strand RNA in infected and transfected cells positive sense primers BL66 59GGATCCAGATCGATCAGC39 PM28 59TGATAAATGGCTTCCTAT39 and BL67 59CCTATTTACATCCTAGG C39 all specific for the heterologous non MHV linker of pB36 Fig 2A were used in seminested reverse transcription PCR RT PCR together with the neg ative sense primer PM112 59CCATGATCAACTTCATTC39 which is comple mentary to nt 18 to 35 of the 39 UTR RNA substrate for structural probing An RNA substrate corresponding to the 39 end of the N gene and almost the entire 39 UTR was transcribed in vitro from SacI truncated plasmid pBL122 Fig 2A Synthesis of uncapped RNA was carried out with an SP6 polymerase transcription kit Ambion per the manu facturer s instructions The resulting transcript was 264 nt long containing 22 vector derived non MHV nt at its 59 end followed by the 39 17 nt of the N gene and 225 nt of the 39 UTR Product RNA was treated with RNase free DNase I Ambion and was purified by extraction twice with phenol chloroform and twice with chloroform followed by two precipitations from ethanol in the presence of 2 M ammonium acetate Prior to enzymatic or chemical probing RNA in the relevant reaction buffer was denatured by incubation at 65 C for 5 min and then allowed to renature by cooling slowly to room temperature in a beaker contain ing 200 ml of H 2 O initially at 65 C Enzymatic structural probing Ten micrograms of synthetic RNA substrate was denatured and renatured in 100 ml of 30 mM Tris HCl pH 7 5 20 mM MgCl 2 300 mM KCl containing 10 mg of yeast tRNA Aliquots 20 ml were then FIG 1 Mutational analysis of the proposed bulged stem loop structure in the MHV 39 UTR 12 Nucleotide numbering begins at the 39 end of the genome excluding the poly A tail the N gene stop codon is boxed The six stem segments of the structure are designated A through F Shown at the right are strand replacement mutants for stem segment D 6912 HSUE ET AL J VIROL on April 13 2015 by UNIV OF CONNECTICUT http jvi asm org Downloaded from incubated at 25 C for 40 min with 10 mg of yeast tRNA and 1 5 10 or 15 U of RNase T 1 Boehringer Mannheim 0 0001 0 001 or 0 01 U of RNase A Boehr inger Mannheim or 0 05 0 1 0 3 or 0 5 U of RNase V 1 Pharmacia 50 Subsequently the enzyme cleaved RNA was subjected to phenol chloroform and chloroform extraction followed by ethanol precipitation prior to primer exten sion analysis 9 51 Chemical modification For modification of RNA with dimethyl sulfate DMS Fluka 10 mg of synthetic RNA was incubated in 200 ml of 80 mM sodium cacodylate pH 7 2 100 mM KCl 5 mM MgCl 2 containing 0 5 1 0 1 5 or 2 0 DMS for 15 min at 25 C 9 21 Reactions were quenched and precipitated by addition of 20 ml of 3 M NaOAc 10 mg of yeast tRNA 500 ml of ethanol For modification of RNA with 1 cyclohexyl 3 2 morpholinoethyl carbodiim ide metho p toluene sulfonate CMCT Aldrich 10 mg of synthetic RNA was incubated in 200 ml of 12 5 mM sodium borate pH 8 1 12 5 mM KCl 2 5 mM MgCl 2 containing 4 2 8 4 12 6 or 16 8 mg of CMCT per ml for 15 min at 25 C 9 21 Reactions were quenched and precipitated by addition of 20 mlof3M NaOAc 10 mg of yeast tRNA 600 ml of ethanol Primer extension analysis Primer PM165 59TCTATCTGTTATGACAGC39 complementary to nt 199 to 216 of the MHV 39 UTR was 59 end labeled with g 32 P ATP using T4 polynucleotide kinase New England Biolabs 21 Purified primer was then annealed to RNase cleaved or chemically modified RNA at 90 C for 3 min and chilled on ice for 5 min Primer extension was carried out in 7 ml reaction mixtures containing 50 mM Tris HCl pH 8 0 50 mM KCl 8 mM MgCl 2 2 mM dithiothreitol 0 85 mM each deoxynucleoside triphosphates and 20 U of avian myeloblastosis virus reverse transcriptase Life Sciences at 42 C for 45 min 51 Samples were separated on a standard 6 0 polyacrylamide DNA sequencing gel containing 8 M urea A sequence ladder was generated directly by dideoxy sequencing of synthetic substrate RNA using the same 59 end labeled primer 20 RESULTS Mutational analysis of each stem of the stem loop structure To facilitate the construction of mutations in the bulged stem loop in the 39 UTR restriction sites adjacent to this structure were designed in pB36 the plasmid used to generate donor RNA for targeted recombination 12 32 Near the upstream boundary we altered codons 450 and 451 of the N gene to create a unique MluI site Fig 2A The first of these changes was coding silent but the second mutated residue 451 of the N protein from aspartate to alanine Based on previous mutagen esis studies this change was expected to be phenotypically silent B Hsue and P S Masters unpublished results Beyond the downstream boundary of the stem loop a single base change was introduced into the same vector creating a unique EcoRV site at nt 219 to 224 of the 39 UTR Fig 2A Here and elsewhere in this paper the coordinates used for the MHV 39 UTR begin at the 39 end of the genome excluding the poly A tail The locus of this change was specifically chosen to not disrupt a proposed downstream pseudoknot structure 56 The resulting plasmid was designated pBL85 To test whether the mutations in pBL85 would have any impact on the replication of MHV targeted RNA recombina tion was carried out to transduce these mutations into the viral genome Fig 2B Donor RNA transcribed from pBL85 was electroporated into cells infected with the thermolabile mutant Alb4 and recombinant viruses were selected as those able to form large plaques at 39 C Two independent recombinants Alb167 and Alb168 were isolated and we confirmed by RNA sequencing that each contained the MluI and EcoRV muta tions from the donor RNA as well as having replaced the 87 nt that were deleted in the Alb4 N gene data not shown Since Alb167 and Alb168 had completely wild type phenotypes this indicated that the mutations created for the two new restric tion sites did not exert any obvious effect on the growth of MHV and thus pBL85 would be an appropriate vector for construction of stem loop mutants In a stepwise manner we next genetically analyzed the func tional role of each stem segment of the bulged stem loop designated A through F Fig 1 To explore whether base pairing or primary sequence of each stem segment plays a role in replication we constructed single arm mutants MAR MBR MBL MCR MDR MDL MER MFR and MFL in which each nucleotide of one arm of a stem segment was changed to its complement thereby disrupting base pairing Fig 1 and 3A To examine whether secondary structure but not primary sequence was important we constructed double arm mutants MALR MBLR MCLR MDLR MELR and MFLR in which the two arms of a stem segment were ex changed thereby restoring the base pairing Fig 1 and 3A In the notation used for these mutants the first letter M or B indicates the origin MHV or BCoV of the remainder of the 39 UTR downstream of the stem loop the second letter A through F indicates the stem segment and the final letters L R or both indicate which arm of the stem segment was re placed The functionality of each of these 15 stem segment muta tions was tested by determining whether it was able to be incorporated into the MHV genome In several independe