An atypical RNA pseudoknot stimulator and an upstream attenuation signal for C01 ribosomal frameshifting of SARS coronavirus Mei Chi Su Chung Te Chang Chiu Hui Chu 1 Ching Hsiu Tsai 1 and Kung Yao Chang Graduate Institute of Biochemistry and 1 Graduate Institute of Biotechnology National Chung Hsing University 250 Kuo Kung Road Taichung 402 Taiwan Received March 7 2005 Revised May 16 2005 Accepted July 6 2005 ABSTRACT The C01 ribosomal frameshifting requires the exist ence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA An atyp ical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus SARS CoV genome This pseudoknot can serve as an efficient stimulator for C01 frameshifting in vitro Mutational analysis of the extra stem suggests frameshift effici ency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus type pseudoknot More importantly an upstream RNA sequence separated by a linker 5 0 to the slippery site is also identified to be capable of modulating the C01 frameshift efficiency RNA sequence containing this attenuation element can downregulate C01 frameshifting promoted by an atypi cal pseudoknot of SARS CoV and two other pseudo knot stimulators Furthermore frameshift efficiency can be reduced to half in the presence of the attenu ation signal in vivo Therefore this in cis RNA attenu ator represents a novel negative determinant of general importance for the regulation ofC01 frameshift efficiency and is thus a potential antiviral target INTRODUCTION The C01 ribosomal frameshifting is a translational regulation mechanism adopted by a variety of viruses to synthesize two or more proteins at a fixed ratio starting with a single trans lation initiation site from the same open reading frame ORF 1 4 In response to the programmed frameshifting signals in messenger RNA mRNA the ribosome is induced to move one base backward in the 5 0 direction and then continues translation in the new C01 reading frame The ratio between the two protein products from frameshifting can be determined by the frameshift efficiency of the stimulation signals and can be a key factor for the propagation of virus within the host It has been suggested to be a potential antiviral target for the interference of viral propagation 5 Efficient induction of eukaryotic C01 ribosomal frameshift ing requires two in cis RNA elements 1 6 The first one is a hepta nucleotides slippery site sequence of X XXY YYZ where the recoding occurs Analysis indicated X can be any three identical nucleotides whereas Y represents three A or U and Z is A U or C In addition a downstream stimulator RNA structure located 6 7 nt away from the slippery site is also needed for efficient frameshifting This downstream RNA stimulator is usually an H type RNA pseudoknot in which nucleotides from a hairpin loop form base pairs with single stranded region outside the hairpin This leads to a topology featuringtwohelicalstems ofbase pairing region stems1 and2 connected by two single stranded loops loops 1 and 2 with a quasi continuous RNA double helical structure However not all RNA pseudoknots can stimulate C01 frameshifting and non pseudoknot RNA element has been characterized to be responsible for inducing C01 frameshifting in HIV 1 7 8 The reported C01 frameshift efficiency induced by different RNA pseudoknots range from 1 to 4 for Beet western yellows virus BWYV in plant cells to 25 and 50 for mouse mammary tumor virus MMTV and avian infectious bronchitis virus IBV respectively in animal cells 6 9 10 There is no clear picture to correlate frameshift efficiency with a specific RNA pseudoknot stimulator However it is thought that resistance of a pseudoknot against deformation by a marching ribosome can cause the elongating ribosome to pause 3 11 13 This will position the A and P site transfer RNAs tRNAs over the slippery site and thus increases the probability for ribosome to slip its A and P site tRNAs in the 5 0 direction by one base and resume the translation in the new reading frame Parameters known to affect frameshift To whom correspondence should be addressed Tel 886 4 22840468 ext 218 Fax 886 4 22853487 Email kychang dragon nchu edu tw C211 The Author 2005 Published by Oxford University Press All rights reserved The online version of this article has been published under an open access model Users are entitled to use reproduce disseminate or display the open access version of this article for non commercial purposes provided that the original authorship is properly and fully attributed the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated For commercial re use please contact journals permissions oupjournals org Nucleic Acids Research 2005 Vol 33 No 13 4265 4275 doi 10 1093 nar gki731 Published online July 29 2005 by guest on November 14 2015 http nar oxfordjournals org Downloaded from efficiency include sequence identity of the slippery site and its distance to the stimulator RNA stability of the stimulator and interactions among the ribosome the mRNA template and the associated tRNAs 1 Recently the sequence identity of the spacer and the E site tRNA have both been shown to modulate frameshift efficiency 14 15 as well In addition host factors have also been implicated to involve in efficiency modulation 16 17 The severe acute respiratory syndrome SARS is an acute respiratory illness caused by a human coronavirus HCV Both SARS CoV and IBV belong to Coronaviridae 18 20 The IBV is known to use a C01 frameshifting mecha nism to generate RNA dependent RNA polymerase RdRp crucial for viral RNA replication The 5 0 portion of the IBV genomic RNA that encodes the RdRp contains a slippery site of the sequences UUUAAAC and is followed by an H type RNA pseudoknot with an in frame stop codon embedded within stem 1 of the folded pseudoknot An elongating ribosome will fall from the viral RNA template without syn thesizing RdRp if C01 frameshifting did not occur in front of this in frame stop codon Therefore defining the biologically relevant mRNA secondary structure which can regulate the C01 frameshift efficiency of the SARS CoV may provide use ful information for anti SARS strategy In this work we report the identification of an atypical RNA pseudoknot of SARS CoV as an efficient RNA stimulator in promoting C01 frameshifting in vitro and explore the role of different stem regions in frameshift efficiency determination In addition we will also demonstrate that particular viral RNA sequences upstream of the slippery site possess an attenuation effect on the overall C01 frameshift efficiency It thus suggests that C01 ribosomal frameshifting can be attenuated by an RNA element upstream of the slippery site in addition to being promoted by a downstream RNA stimulator MATERIALS AND METHODS Construction of reporter genes and mutagenesis Plasmid encoding the gene for ORF 1ab junction region of SARS CoV pCRII SARS 12 265 13 653 was a gift from Professor Pei Jer Chen and was used as the template for PCR cloning of the cDNA of viral RNA fragments The p2luc reporter was a gift from Professor John Atkins at the University of Utah 21 and the pRL SV40 vector was purchased from Promega Forward and reverse DNA primers respectively carrying the SalI and BamHI restriction sites and appropriately designed annealing sequences were used for PCR amplifica tion of the desired cDNA encoding SARS CoV viral RNAs from pCRII SARS 12 265 13 653 The amplified inserts were then sub cloned into the SalI BamHI sites of p2luc using standard procedures and the resulting recombinant vectors were trans formed into DH5a cell for the maintenance and selection by ampicillin Frameshifting stimulator sequences of the minimal IBV and MMTV pseudoknot were chemically synthesized They were amplified by forward and reverse primers respect ively containing BsrGI and BsaAI site and ligated into the BsrGI BsaAI site 1392 1426 of restriction enzymes treated pRL SV40 vectors All of the base pairing disruption and restoration mutants were constructed by using the quick change mutagenesis kit from Stratagene according to the manufacturer s instructions In contrast PCR based ligation approach with appropriate primers was used to assemble different chimera which carried the attenuation signal the sequences are available upon request and will be described briefly Initially two comple mentary oligonucleotides containing the sense and the anti sense sequences corresponding to the 3 0 end and 5 0 star region of the two fragments planned to be jointed were synthesized chemically They were then used as forward sense or reverse antisense primers in combination with the corresponding reverse or forward primer carrying appropriate restriction sites to amplify the desired pre jointed fragments from pCRII SARS 12 265 13 653 The two individual PCR products with partially overlapping sequences were then assembled by the PCR based ligation procedure 22 and then sub cloned into the BamHI EcoRI sites of p2luc vectors The identities of all cloned and mutated genes were confirmed by DNA sequencing analysis RNA structure probing RNA transcripts spanning the SARS 13 377 13 475 region of SARS CoV were generated by in vitro transcription using T7 RNA polymerase The purified RNA of desired length was then dephosphorylated by shrimp alkaline phosphatase and 5 0 end labeled with g 32 P ATP using T4 polynucleotide kinase and separated by a 12 sequencing gel All the RNase protection experiments were performed in 50 ml reaction vol umes containing 50 000 70 000 c p m of 5 0 end labeled RNA in the presence of RNase cleavage buffer 30 mM Tris HCl pH 7 5 3 mM EDTA and 200 mM NaCl and 10 mM MgCl 2 was included in the same buffer for RNase V1 experiments Before the addition of probing enzymes the RNAs were denat ured by heating at 70 C14 C for 5 min followed by instant cooling on ice and brought back to 20 C14 C for structural mapping The following amounts of RNases were added for each reaction 0 0088 1000 0 044 200 mg RNase A USB 0 05 10 U RNase T1 USB 0 5 15 U RNase T2 USB and 0 01 01 U RNase V1 Amersham Pharmacia The hydrolysis RNA lad ders were obtained by the incubation of RNA in the hydrolysis buffer at 85 C14 C for 10 min and parallel RNA sequencing prod ucts were obtained by the treatment of unfolded RNA with RNases T1 and A They were used as markers for the assign ment of guanines and pyrimidines respectively All the reac tions were incubated at 20 C14 C for 10 min with the exception that RNase V1 was incubated for 15 min The reactions were terminated by phenol chloroform extraction and precipitated washed with 70 ethanol and dried by vacuum Finally the cleavage products were loaded into a 10 denaturing gel with different running time to resolve different parts of RNA and visualized by using phosphorimagery In vitro transcription translation and frameshift assay The T7 coupled transcription translation TNT system Progema was used for the generation of the shifted and the non shifted protein products according to the manufac turer s instructions In each assay a 25 ml reaction containing 500 ng of DNA template 12 5 ml of reticulocyte lysate and 0 8 mlof10mCi 35 S labeled methionine NEN was incubated at 30 C14 C for 1 5 h The samples were then resolved by 12 SDS PAGE and exposed to PhosphorImager screen 4266 Nucleic Acids Research 2005 Vol 33 No 13 by guest on November 14 2015 http nar oxfordjournals org Downloaded from for quantification after drying The frameshift efficiency was calculated by dividing the counts of the shifted product by sum of the counts for both shifted and non shifted products with correction of the methionine number in each product Control experiments with the plasmids of 100 1000 ng produce similar results for frameshift efficiency data not shown For the generation of capped mRNA templates the mMESSAGE mMACHINE kit Ambion was used according to the manufacturer s instructions Mammalian cell culture and luciferase assay Human embryonic kidney HEK 293T cells were cultured in the DMEM supplemented with 10 fetal bovine serum The transient expression was performed by LIPOFECTIN Gibco BRL transfection of 500 ng of reporter plasmids into 12 wells cultured cells The cells were then assayed for the transient expression of the reporter gene 24 h after transfection using dual luciferase assay of cell lysates and normalized by b galactosidase activity as an internal control All the in vivo experiments were repeated three times with four to six assays for each reaction Luciferase activity measure ments for both in vitro reticulocyte lysate and in vivo trans fected 293T cell lysates were performed by using the dual luciferaseC212 reporter assay Promega according to the manufacturer s instructions on an ABI TR 717 luminometer Calculation of luciferase based frameshift efficiency was performed with control plasmids to calibrate the ribosome drop off effect as described previously 21 RESULTS The C01 frameshifting stimulator of SARS CoV contains an atypical H type pseudoknot with an extra stem loop in loop 2 Sequence alignment of the junction regions of ORF 1ab of SARS CoV and other coronaviruses indicated the existence of conserved UUUAAAC slippery site followed by a stretch of viral RNA sequences with the potential to form an H type RNA pseudoknot Figure 1 This region of the SARS CoV RNA genome SARS 13 369 13 520 for residues 13 369 13 520 was thus cloned into p2luc vector to examine its C01 frameshifting activity 21 To faithfully monitor frameshift efficiency we also arranged the terminus of the viral sequences under investigation to keep the TAA sequences in the N terminal part of firefly luciferase ORF in frame to act as a stop codon in the presence of C01 frameshifting This leads to premature termination of the frameshifted product and thus prevents the underestimation of frameshift effici ency because this manipulation can minimize the ribosome drop off effect while translating the full length firefly ORF 21 A different vector pRL SV40 was also used as the second reporter Frameshift assay using either of the reporters indicated that the selected viral RNA sequences pos sess a frameshift efficiency of 60 data not shown which suggests that the SARS 13 369 13 520 RNA contains an efficient C01 frameshifting stimulator in vitro Further examination of sequence contents of the SARS 13 369 13 520 RNA revealed a potential stem loop struct ure within the loop 2 region of this IBV type pseudoknot Furthermore their primary sequences were found to be con served among isolated SARS CoV sequences and similar stem loop structures were also identified in the loop 2 region of bovine coronavirus and mouse hepatitis virus pseudoknot Figure 1 To confirm the existence of the predicted atypical pseudoknot enzymatic structure probing experiments were performed on SARS 13 377 13 475 RNA and some of the results are shown in Figure 2A As can be seen the distribution of cleavage patterns for ribonuclease probes sensitive to single strand region such as RNase T2 T1 and A in lanes 7 12 of Figure 2A is in agreement with the existence of loop regions whereas the cleavage pattern by RNase V1 lanes 5 and 6 that prefers cutting of duplex and stacked conformations can be localized to the three predicted stem regions S1 S2 and S3 However co existence of cleavage by both RNase V1 and single strand sensitive probes can be found for nucleotides localized to the junction region between S2 and S3 This is probably caused by a dynamic conformational equilibrium in this region as non denaturing gel analysis suggests that the Figure 1 Characterization of the in cis RNA elements involved in C01 frameshifting for SARS CoV Sequence alignment for a set of related coronaviruses The slippery site is boxed and typed in gray whereas the complementary base pairing counterpart of stem regions 1 and 2 S1 S1C and S2 S2C of stimulator are underlined and boxed by solid line respectively The potential base pairing scheme for the third stem S3 is underlined by dashed line The S2C region for HCV 229E is not shown as it appears in further downstream region Nucleic Acids Research 2005 Vol 33 No 13 4267 by guest on November 14 2015 http nar oxfordjournals org Downloaded from A B 4268 Nucleic Acids Research 2005 Vol 33 No 13 by guest on November 14 2015 http nar oxfordjournals org Downloaded from SARS 13 377 13 475 RNA adopts a single conformation data not shown Nevertheless these probing data are consistent with the existence of a stem conformation within the loop 2 in this atypical IBV type pseudoknot as summarized in Figure 2B Recently the same S3 conformation has also been proposed and verified independently 23 24 Disruption of the base pairing region in S3 of an atypical pseudoknot impairs C01 frameshifting stimulation activity of the SARS 13 369 13 520 RNA To define the contribution of dif ferent stem regions of this atypical IBV type pseudoknot in the stimulation of C01 frameshifting activity we mutated those nucleotides mapped onto form the duplex conformations Mutants expected to disrupt a stem and those expect to reform the stem by compensatory base changes were then measured for their efficiency in promoting C01 frameshifting in vitro Figure 3A As shown in lanes 1 3 of Figure 3B frameshift efficiency drops from 60 for wild type construct to 1 for both 5 0 and 3 0 mutants that prevent the formation of base pairing in S1 In contrast frameshift efficiency for the mutant with compensatory base changes to restore the base pairing scheme in S1 lane 2 of Figure 3C rises back to the wild type level 55 Similar results are observed for S2 with mutants destroying or reforming base pairs in the mapped duplex region lanes 4 and 5 of Figure 3B and Lane 3 of Figure 3C These experiments complement structural map ping data and strongly support the idea that it is the formation of the two typical H type pseudoknot stems S1 and S2 and not their sequence identities are required for efficient C01 frameshifting stimulation Interestingly lower sensitivity to base pairing disruption is observed for mutants located in the mapped S3 region Mutants with disruption of base pairing in the upper or and in the lower stem region of S3 both led to a substantial decrease in frameshift efficiency when compared with the wild type construct from 60 to 36 or 37 respectively in lanes 6 and 7 of Figure 3B However restoring the base pairing scheme in either portion by compensatory base changes restored frameshift efficiency to the wild type level including the up restoration mutant that even displays a com pletely different sequence composition lanes 4 and 5 in Figure 3C suggesting that the formation of base pairing and not the sequence contents in S3 is involved in the efficient frameshifting activity of this atypical IBV type pseudoknot Intriguingly frameshift efficiency does not reduce further when more bases were mutated to disrupt all the potential base pair in S3 lanes 8 and 9 of Figure 3B implicating the existence of a residual frameshift activity for the remaining pseudoknot scaffold Furthermore a pseudoknot mutant lack ing the S3 but harboring an 8 nt loop 2 possesses a frameshift efficiency of 53 lane 10 in Figure 3B As the topology and activity of this mutant are similar to those of the minimal IBV pseudoknot that lacks 75 of its wild type loop 2 sequences 25 which indicates that S3 is not absolutely required for high frameshift efficiency To rule out the possibility that the observed frameshift efficiency variation in S3 mutants is the outcome of their differences in ribosome loading efficiency of uncapped