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and al 77843 2 Keywords Coronavirus or these compensatory Mutations opening up S3 were lethal Disruptions of S4 generated both viable and lethal mutants Genomes carrying the original mutations in S3 or S4 plus compensatory mutations restoring base pairing positive sense b in le int Siddell vely studied structure 171 nts of the 1995b the secondary 68 nts of the 3 viral replication supported this it was necessary and the rst 1997 Williams et al Williams et al 1999 examined the 3 UTR in Contents lists available at SciVerse ScienceDirect journal homepage Virology leibowitz medicine tamhsc edu J L Leibowitz Virology 443 2013 40 47 the 5 direction in MHV and its 5 stem overlaps with the lastUSA bovine coronavirus and reported evidence for a phylogenetically conserved pseudoknot This pseudoknot encompasses nts 238 185 note that the 3 most nucleotide upstream of the poly A tail is position 1 in this numbering scheme with numbers ascending in 0042 6822 see front matter Hsue and Masters n Corresponding authors E mail addresses pliu tmhs org P Liu genomic RNA and six to seven subgenomic mRNAs that make up a 3 co terminal nested set Leibowitz et al 1981 van der Born 2008 Analysis of deletion mutants of MHV defective interfering DI RNAs indicated that DI replication requires cis acting elements located within the 3 terminal 436 nucleotides of the virus Kim et al 1993 Lin and Lai 1993 This 436 nts region extends upstream of the 301 nts 3 UTR into the nucleocapsid N protein gene Subsequent studies demonstrated that in the context of the entire virus the N protein Williams et al 1999 Yu and Leibowitz 1995a 1 2008 Initial studies of the MHV 3 UTR RNA secondary predicted three stem loop structures in the 3 most genome excluding the poly A tail Yu and Leibowitz Subsequently the Hsue and Masters also examined structure of the 3 UTR and predicted that the 5 UTR folded into a bulged stem loop required for Hsue and Masters 1997 Biochemical studies bulged stem loop structure and suggested that for RNA replication although the terminal loop 32 kb betacoronavirus Britton 2008 Cells infected with MHV contain Hsue and Masters 1997 Johnson et al 2005 Liu et al 2001 1997 995b Zust et al 3 UTR RNA secondary structure MHV Mutation Reverse genetics Introduction Coronaviruses are single stranded contain a genomic RNA from 26 32 k 1997 Coronaviruses have been divided antigenic and sequence comparisons virus MHV one of the most extensi Goebel et al 2004 Multiple studies have been performed to determine the RNA secondary structures present in the 3 UTR and their functional role in viral replication Goebel et al 2004 2007 Hsue et al 2000 Cis acting elements were viable and had robust growth phenotypes These results support the Zust model for the coronavirus 3 UTR and suggest that the S3 stem is required for virus viability Functional analysis of the stem loop S3 coronavirus 3 UTR Pinghua Liu a n 1 Dong Yang a Kristen Carter b 2 Fary a Department of Microbial and Molecular Pathogenesis Texas A viruses containing the ATW3 and ATW5 mutations grew almost as well as wild type virus in a one step growth curve experiment although they did produce signi cantly smaller pla ques than wild type Johnson et al 2005 Goebel et al Goebel et al 2007 demonstrated that a hyper variable bulged stem loop spanning nts 46 156 was not essential for MHV replication even though this otherwise poorly conserved region contains a highly conserved octanucleotide sequence present in virtually all coro naviruses Boursnell et al 1985 and proposed a secondary structure model of the complete MHV 3 UTR Goebel et al 2004 Goebel et al 2007 Based in part on sequence co variation Zust et al Zust et al 2008 subsequently revised the 3 region of this model see Fig 1 tore ect potential conserved base pairings present in viruses representing all three coronavirus subgroups Although the phylogenetic conservation of the proposed second ary structural model is persuasive and suggests that the proposed secondary structures are functionally important a genetic test of the functional importance of this structure was not performed We report here a functional analysis of the RNA secondary structure model proposed by Zust et al Zust et al 2008 We utilized reverse genetic approaches to experimentally test this model through mutagenesis of the novel S3 and S4 stems see Fig 1 predicted by this model Mutations opening up S3 were lethal but large disruptions in S4 generated both viable and lethal mutants Genomes carrying the original mutations in S3 or S4 plus compensatory mutations that restored base pairing in these stems were all viable and had robust growth phenotypes Overall our results support the Zust model for the coronavirus 3 UTR and suggest that the S3 stem is required for virus viability whereas at least some mutations that disrupt the S4 stem can be tolerated Results Small disruptions of S3 or S4 have little effect on viral phenotype and negative strand subgenomic RNA synthesis In the Zust et al model of the MHV 3 UTR Zust et al 2008 nucleotides 0 to 9 note that the 5 most A of the poly A tail is designated as position 0 are base paired with the L1 loop of the pseudoknot to form a helical stem labeled S3 in Fig 1A nucleotides 18 29 are base paired S4 in Fig 1A with sequences downstream of the pseudoknot stem S2 In an earlier study of protein interacting sequences in the 3 UTR Johnson et al 2005 three mutations that segment of the upstream bulged stem loop positions 301 224 in MHV making the two secondary structures in part mutually exclusive This led to the hypothesis that these two structures may be alternate conformers of the same region of genomic RNA and constitute a molecular switch Hsue et al 2000 A genetic analysis using viral mutants isolated by targeted recombination supports this hypothesis Goebel et al 2004 A study examining the secondary structure of the last 166 nucleotides of the 3 UTR a region downstream of the pseudoknot predicted a long multi branch stem loop in this region of the genome a model that was largely supported by enzymatic probing of RNA secondary struc ture Liu et al 2001 DI RNA replication assays supported a mapped to S3 and or S4 see Fig 1 in the Zust et al model of the MHV 3 UTR Zust et al 2008 were examined for their effects on replication These mutations originally named ATW3 ATW5 andATW Johnson et al 2005 have been renamed as A B and AB in the current study to increase clarity see Fig 1B and C When these three mutations are introduced into the genomic RNA genomes containing the A and B mutations lead to viable viruses however the genome containing the AB mutation is lethal Johnson et al 2005 Based on our earlier results with the A B and AB mutants we hypothesized that the presence of either of the two stems is suf cient for a viable virus but disruption of both stems is lethal To investigate this hypothesis and to perform a genetic test of the Zust secondary structure model of the 3 UTR we made a series of additional mutations targeting S3 and S4 Mutations C and D were designed to destabilize S3 and S4 respectively mutation CD destabilizes both S3 and S4 Fig 1B Mutations that affected both sides of S3 and S4 were designated ABD ABC and ABCD Fig 1C WhenmutationsC D ABC andABD whichdestabilizebase pairingin either the S3 D ABC or S4 C ABD helices were introduced into the genomic RNA they all produced viable virus with somewhat smaller plaque sizes compared to the wild type virus Fig 2A grew with near identical kinetics to wild type virus and achieved peak titers that were at least 25 of those achieved by wild type virus Fig 2B However for genomes carrying mutation CD a mutation which disrupts base pairing in both S3 and S4 we were unable to recover virus in multiple trials thus we concluded that this mutation was lethal similar to the resultsweobtainedwiththeA B andABmutationsinourprevious work Johnson et al 2005 Furthermore when mutant ABCD which restores base pairing in stems S3 and S4 but alters the sequences of these stems was introduced into the genomic RNA it was viable and generated a virus that forms plaques almost identical in size to those formed by wild type virus grew with nearly identical kinetics to wild type virus and achieved a peak titer nearly identical to that of wild type virus Fig 2A and B These results are entirely consistent with the Zust model Zust et al 2008 ofthe3 UTR and our hypothesis that base pairing in either S3 or S4 are necessary for MHV viability We had previously demonstrated that MHV genomes carrying the lethal AB mutation are unable to direct the synthesis of subgenomic RNA although they are able to direct the synthesis of a minus sense complement of the genome Johnson et al 2005 To determine the RNA species that might have been generated in cells electroporated with genomes containing the CD mutation we performed nested RT PCR assays to detect negative strand genomic RNA and negative strand subgenomic RNA3 and mRNA6 These RNAs serve as templates for genomic and subgenomic mRNA synthesis Pasternak et al 2003 Sawicki and Sawicki 1990 Sola et al 2005 Zuniga et al 2004 Nested RT PCR results showed that negative strand genomic RNAs were present in cells at 4 and 8 h after electroporation with in vitro transcribed genomes carrying the CD mutation similar to what we observed after electroporation of WT genomes Fig 2C In contrast neither negative strand subgenomic RNA3 nor RNA6 were detected in cells electroporated with in vitro transcribed genomes carrying the CD mutation whereas cells electroporated with WT genomes contained negative strand subgenomic RNA3 and RNA6 at 8 h incubation Fig 2D and E For each sample parallel RT PCR reactions without an RT step were performed to ensure that residual DNA templates taken up by the cells during electroporation did not produce PCR signals data not shown These data show that genomes carrying the CD mutation destabilizing stems S3 and S4 are defective in directing subgenomic RNA synthesis the identical phenotype detected with the AB mutations on the opposing sides of the stem in our previous work Johnson et al 2005 Larger disruptions of S3 or S4 generate various viral phenotypes Based on the above results we further tested if larger sequence disruptions in S3 or S4 affect virus viability A series of mutants E F G H EH and FG were made for this purpose Fig 3 Mutations E P Liu et al Virology 443 2013 40 4742 and H two mutants that completely disrupt S3 were lethal a mutation in the 5 side of S4 that completely disrupted this stem mutation G produced the same lethal phenotype However the mutation in the 3 side of S4 mutation F produced a viable virus All plaque isolates of F mutant virus from two independent electroporations also contained a second site mutation either A5 C5 or A6 C6 C5 or C6 can base pair with G221 which is extruded in the WT S3 stem this base pairing increased the stability of S3 in viruses we recovered containing the F mutation Sequencing of the nsp8 and nsp9 coding regions of these mutants failed to reveal additional second site mutations Unsurprisingly mutations that restored the S3 and S4 helices mutations EH and FG both generated viable mutants viruses EH and FG These viable mutants have smaller plaque sizes but essentially equivalent one step growth curve compared to wild type virus Fig 4A and C RNA species present in cells electroporated with the lethal G and H mutants were analyzed by RT PCR as described above for the AB and CD lethal mutants negative sense genomic RNAs were detected when cells were electroporated with genomes containing the lethal mutants H in S3 and G in S4 however subgenomic RNA Fig 1 Proposed RNA secondary structures and mutations in the MHV 3 UTR A 3 UTR comprised of the HVR and the two helical stem structures S3 and S4 B Mutations made the A and B were previously made by Johnson et al 2005 The wild type sequence is shown sequence of each mutant C Mutated nucleotides are indicated by italicized lower case synthesis is defective in these mutants Fig 4D and E These results suggest that S3 is critical for virus viability with complete disruption of S3 leading to a defect in subgenomic RNA synthesis and thus a lethal phenotype The disparate effects of the F viable G lethal and FG viable mutations in S4 led us to model the possible effects of these three mutations on the overall folding of this portion of the 3 UTR in Mfold The Mfold models suggest that the G mutant has the potential to fold into a very different structure than the wild type structure but with a similar thermo dynamic stability perhaps accounting for the G mutant s lethal phenotype In contrast the F mutation is predicted to simply result in local unfolding of S4 This suggests that S4 is not essential for virus viability Disruption of L3 has no effect on viral phenotypes The 8 nts that make up loop 3 L3 see Fig 1AandFig 3 havea perfect palindromic sequence arrangement and led us to investi gate the role of L3 if any in viral replication Three mutations a loop randomized mutation LRD a mutation that shortened L3 by model proposed by Zust et al 2008 including the region downstream of S2 to locally disrupt S3 and S4 The C and D mutations were made in this study and and the nucleotides that are altered by mutation italicized Arrows indicate the letters P Liu et al Virology 443 2013 40 47 43 2 nts LST2 and a mutation that shortened L3 by 6 nts LST6 were designed to test the functional role of L3 Fig 3 Interestingly these mutations all resulted in viable viruses with only minor differences in plaque size and no differences in their replication kinetics or nal titer compared to wild type virus Fig 4B and C This result showed that L3 likely functions as a linker between S3 and S4 with few constraints on its sequence and length Discussion Cis acting sequences important for controlling viral replication are typically found in the 3 and 5 UTRs of RNA viruses RNA Fig 2 Growth phenotypes for viable mutant viruses and RNA phenotypes for lethal mutant PCR assays for negative sense RNA corresponding to genomic RNA D subgenomic RNA3 represent RNA extracted from cells that were mock electroporated with buffer rather than RNA species are indicated by arrows secondary structural features are often important in the function of these cis acting regions In this study we have performed a functional analysis of the RNA secondary structural model of the MHV 3 UTR originally proposed by Zust et al Zust et al 2008 utilizing a reverse genetics approach The conservation amongst group 2 coronaviruses of the structural elements of the 3 UTR namely the bulged stem loop and overlapping pseudoknot and the conserved S3 and S4 helices connected by a variable length loop see Fig 1A Zust et al 2008 and the ability of other group 2 coronavirus SARS CoV and BCoV 3 UTRs with primary sequences that diverge signi cantly from MHV to functionally replace the MHV 3 UTR Goebel et al 2004 Hsue and Masters 1997 Kang et al 2006 suggests the RNA secondary structure of A Plaque size mm of viable viruses B Growth curve of viable viruses C RT and E RNA6 for wild type MHV A59 and lethal mutant CD Lanes labeled Negative with in vitro transcribed RNA The positions of the expected PCR products for each P Liu et al Virology 443 2013 40 4744 the 3 UTR plays a key functional role in coronavirus replication The overlap of the most distal portion of the bulged stem loop with the pseudoknot makes these two structures mutually exclu sive This led to the hypothesis that these two structures may be alternate conformations of the same region of genomic RNA and constitute a molecular switch Hsue et al 2000 Genetic studies demonstrated that both the pseudoknot and base pairing of the lower region of the bulged stem loop were functionally important supporting this hypothesis Goebel et al 2004 Bio physical studies Stammler et al 2011 demonstrate that the pseudoknotted conformation is much less stable than the double hairpin conformation but suggest that stacking of the pseudoknot with the S3 helix can stabilize the pseudoknotted conformation allowing it to form Mutations predicted to disrupt the S3 helix mutations E and H were both lethal whereas the double muta tion EH predicted to restore the S3 helix resulted in a viable virus consistent with the idea that this stem may be necessary to allow the pseudoknotted conformation to form or is necessary for viral replication in its own right Mutations in S4 predicted to disrupt this helix had differing effects Mutation G in the 5 side of S4 and predicted to completely disrupt the S4 helix produced a lethal phenotype This contrasted with the mutation in the 3 side of the S4 helix mutation F which reproducibly produced a viable virus with a second site mutation in the S3 helix that stabilized this structure The double mutant FG predicted to restore the predicted structure resulted in a viable Fig 3 Mutations causing complete disruption of S3 or S4 helices or the L3 virus entirely consistent with the Zust Zust et al 2008 model Modeling the possible effects of these three mutations on the overall folding of this portion of the 3 UTR in Mfold suggests that the G mutant has the potential to cause the region containing the triple helix junction S2 S3 and S4 see Fig 1 to fold into a completely different two helix structure with a similar thermo dynamic stability as the wild type structure This structure lacking the S2 stem loop and both the S3 and S4 helices would not be able to form the pseudoknotted structure or the double stem loop structure extended bulged stem loop plus S2 L2 stem loop likely accounting for the G mutant s lethal phenotype In contrast the F mutation is predicted to simply result in local unfolding of S4 All of the viruses we recovered with the F mutation in S4 also contained second site A5C or A6C mutations in S3 that resulted in aG C base pair replacing an A U base pair thus increasing in the stability of S3 by a small amount Our failure to recover viruses that contain the F mutation without any second site mutations raises the possibility that S4 interacts stacks with the S3 helix making a further necessary contribution to the stability of the 3 UTR particularly in the pseudoknotted con guration Mutations with smaller sequence disruptions designed to sepa rately destabilize the predicted S3 mutations A D ABC or S4 mutations B C and ABD helical stems were all viable whereas mutants that destabilized both of these helices mutations AB and CD were lethal A quadruple mutation ABCD that maintained both S3 and S4 while altering the sequences of these stems yielded a virus loop Mutated nucleotides are indicated by italicized lower case letters P Liu et al Virology 443 2013 40 47 45 with an in vitro phenotype virtually identical to that of wild type virus These results strongly support the Zust Zust et al 2008 model Modeling the possible effects of the lethal AB and CD mutations on the overall folding of this portion of the 3 UTR in Mfold suggests that the CD mutant has only one thermodynamically stable conformation in which the region containing the triple helix junction S2 S3 and S4 see Fig 1 folds into a completely different structure consisting of a long bulged stem loop linked to a shorter bulged stem loop by a seven nucleotide single stranded linker This structure lacking the S2 L2 stem loop and both the S3 and S4 helices would not be able to form the pseudoknotted structure or the alternative two stem loop struc ture that make up the putative molecular switch Goebel et al 2004 possibly accounting for the CD mutant s lethal phenotype It is less clear from our modeling of the AB mutant why it is lethal The three structures predicted by Mfold all maintain the S2 L2 stem loop with the most stable predicted structure containing an additional bulge in S4 and a somewhat recon gured and shorter S3 helix see Fig 1 These same altered S3 and S4 helices are predicted to be present singly in the A and B mutants