CORD-19-Sentences  

CORD-19:798f848bfe9a46c23560c06984aab9637c08c907 JSONTXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE-old TextAE

Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-66 Sentence denotes Programmed ribosomal frameshifting in decoding the SARS-CoV genome
TextSentencer_T1 0-66 Sentence denotes Programmed ribosomal frameshifting in decoding the SARS-CoV genome
TextSentencer_T2 68-76 Sentence denotes Abstract
TextSentencer_T2 68-76 Sentence denotes Abstract
TextSentencer_T3 77-188 Sentence denotes Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses.
TextSentencer_T3 77-188 Sentence denotes Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses.
TextSentencer_T4 189-387 Sentence denotes Comparative analysis of the frameshift region reveals a universal shift site U _ UUA _ AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops.
TextSentencer_T4 189-387 Sentence denotes Comparative analysis of the frameshift region reveals a universal shift site U _ UUA _ AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops.
TextSentencer_T5 388-527 Sentence denotes Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry.
TextSentencer_T5 388-527 Sentence denotes Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry.
TextSentencer_T6 528-698 Sentence denotes Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U _ UUA _ AAC.
TextSentencer_T6 528-698 Sentence denotes Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U _ UUA _ AAC.
TextSentencer_T7 699-947 Sentence denotes Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation.
TextSentencer_T7 699-947 Sentence denotes Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation.
TextSentencer_T8 948-1096 Sentence denotes These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.
TextSentencer_T8 948-1096 Sentence denotes These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.
TextSentencer_T9 1097-1098 Sentence denotes D
TextSentencer_T9 1097-1098 Sentence denotes D
TextSentencer_T10 1100-1243 Sentence denotes Directed switching of a proportion of translating ribosomes into a new reading frame results in the synthesis of a trans-frame protein product.
TextSentencer_T10 1100-1243 Sentence denotes Directed switching of a proportion of translating ribosomes into a new reading frame results in the synthesis of a trans-frame protein product.
TextSentencer_T11 1244-1434 Sentence denotes This programmed ribosomal frameshifting is an essential mechanism governing expression of a subset of viral and cellular genes (Baranov et al., 2003; Namy et al., 2004; Stahl et al., 2002) .
TextSentencer_T11 1244-1434 Sentence denotes This programmed ribosomal frameshifting is an essential mechanism governing expression of a subset of viral and cellular genes (Baranov et al., 2003; Namy et al., 2004; Stahl et al., 2002) .
TextSentencer_T12 1435-1610 Sentence denotes One example is in coronaviruses, where a fixed portion of the ribosomes translating orf1a change reading frame at a specific location to decode information contained in orf1b.
TextSentencer_T12 1435-1610 Sentence denotes One example is in coronaviruses, where a fixed portion of the ribosomes translating orf1a change reading frame at a specific location to decode information contained in orf1b.
TextSentencer_T13 1611-1806 Sentence denotes This translational event is essential for the synthesis of viral RNA-dependent RNA polymerase and other replication components, as orf1b lacks its own independent site for translation initiation.
TextSentencer_T13 1611-1806 Sentence denotes This translational event is essential for the synthesis of viral RNA-dependent RNA polymerase and other replication components, as orf1b lacks its own independent site for translation initiation.
TextSentencer_T14 1807-1944 Sentence denotes This simple mechanism for gene expression ensures that product of orf1b is expressed at specific levels relative to the product of orf1a.
TextSentencer_T14 1807-1944 Sentence denotes This simple mechanism for gene expression ensures that product of orf1b is expressed at specific levels relative to the product of orf1a.
TextSentencer_T15 1945-2067 Sentence denotes Frameshifting in coronaviruses was first demonstrated in Avian Infectious Bronchitis Virus (IBV) (Brierley et al., 1989) .
TextSentencer_T15 1945-2067 Sentence denotes Frameshifting in coronaviruses was first demonstrated in Avian Infectious Bronchitis Virus (IBV) (Brierley et al., 1989) .
TextSentencer_T16 2068-2397 Sentence denotes Extensive pioneering analysis by Brierley et al. (1991 Brierley et al. ( , 1992 , Liphardt et al. (1999) , and Napthine et al. (1999) discovered that ribosomes shift translation frame at the slippery sequence U _ UUA _ AAC, which is invariant among known coronaviruses, and is stimulated by a downstream RNA pseudoknot structure.
TextSentencer_T16 2068-2397 Sentence denotes Extensive pioneering analysis by Brierley et al. (1991 Brierley et al. ( , 1992 , Liphardt et al. (1999) , and Napthine et al. (1999) discovered that ribosomes shift translation frame at the slippery sequence U _ UUA _ AAC, which is invariant among known coronaviruses, and is stimulated by a downstream RNA pseudoknot structure.
TextSentencer_T17 2398-2524 Sentence denotes As predicted (Ten Dam et al., 1990) , 3VRNA pseudoknots are widely used for stimulating 1 programmed frameshifting in viruses.
TextSentencer_T17 2398-2524 Sentence denotes As predicted (Ten Dam et al., 1990) , 3VRNA pseudoknots are widely used for stimulating 1 programmed frameshifting in viruses.
TextSentencer_T18 2525-2719 Sentence denotes Mutagenic and structural data for several of the frameshift stimulators point to key elements required for their activity [For reviews, see: (Brierley and Pennell, 2001; Giedroc et al., 2000) ].
TextSentencer_T18 2525-2719 Sentence denotes Mutagenic and structural data for several of the frameshift stimulators point to key elements required for their activity [For reviews, see: (Brierley and Pennell, 2001; Giedroc et al., 2000) ].
TextSentencer_T19 2720-2904 Sentence denotes Significant differences in these key elements suggest that multiple forms of pseudoknots with differences in both secondary and ternary structures can be active frameshift stimulators.
TextSentencer_T19 2720-2904 Sentence denotes Significant differences in these key elements suggest that multiple forms of pseudoknots with differences in both secondary and ternary structures can be active frameshift stimulators.
TextSentencer_T20 2905-3028 Sentence denotes For the Mouse Mammary Tumor Virus (MMTV) pseudoknot, there is a single unpaired adenine that separates stem I from stem II.
TextSentencer_T20 2905-3028 Sentence denotes For the Mouse Mammary Tumor Virus (MMTV) pseudoknot, there is a single unpaired adenine that separates stem I from stem II.
TextSentencer_T21 3029-3145 Sentence denotes This adenine is reported to be responsible for a bend between the 0042-6822/$ -see front matter D 2004 Elsevier Inc.
TextSentencer_T21 3029-3145 Sentence denotes This adenine is reported to be responsible for a bend between the 0042-6822/$ -see front matter D 2004 Elsevier Inc.
TextSentencer_T22 3146-3412 Sentence denotes All rights reserved. doi:10.1016/j.virol.2004.11.038 two stems and it has been proposed that this adenine and the resulting bent conformation are essential for frameshift stimulation (Chen et al., 1995 (Chen et al., , 1996 Kang and Tinoco, 1997; Kang et al., 1996) .
TextSentencer_T22 3146-3412 Sentence denotes All rights reserved. doi:10.1016/j.virol.2004.11.038 two stems and it has been proposed that this adenine and the resulting bent conformation are essential for frameshift stimulation (Chen et al., 1995 (Chen et al., , 1996 Kang and Tinoco, 1997; Kang et al., 1996) .
TextSentencer_T23 3413-3529 Sentence denotes Similarly, there is a single unpaired adenine between stems I and II in simian retrovirus type-1 (SRV-1) pseudoknot.
TextSentencer_T23 3413-3529 Sentence denotes Similarly, there is a single unpaired adenine between stems I and II in simian retrovirus type-1 (SRV-1) pseudoknot.
TextSentencer_T24 3530-3758 Sentence denotes However, unlike MMTV, altering the nucleotide in this position and the corresponding base across the stem to be either paired or unpaired does not have a dramatic effect on the efficiency of frameshifting (Sung and Kang, 1998) .
TextSentencer_T24 3530-3758 Sentence denotes However, unlike MMTV, altering the nucleotide in this position and the corresponding base across the stem to be either paired or unpaired does not have a dramatic effect on the efficiency of frameshifting (Sung and Kang, 1998) .
TextSentencer_T25 3759-3891 Sentence denotes The crystal structure of the Beet Western Yellow Virus (BWYV) frameshift pseudoknot reveals several significant structural features.
TextSentencer_T25 3759-3891 Sentence denotes The crystal structure of the Beet Western Yellow Virus (BWYV) frameshift pseudoknot reveals several significant structural features.
TextSentencer_T26 3892-4058 Sentence denotes Notably, adenine-rich loop II forms extensive hydrogen bond interactions with stem I, and a base-triple is formed between an unpaired cytosine in loop II and stem I .
TextSentencer_T26 3892-4058 Sentence denotes Notably, adenine-rich loop II forms extensive hydrogen bond interactions with stem I, and a base-triple is formed between an unpaired cytosine in loop II and stem I .
TextSentencer_T27 4059-4176 Sentence denotes Mutagenic studies demonstrated that these interactions are critical for frameshifting efficiency (Kim et al., 1999) .
TextSentencer_T27 4059-4176 Sentence denotes Mutagenic studies demonstrated that these interactions are critical for frameshifting efficiency (Kim et al., 1999) .
TextSentencer_T28 4177-4353 Sentence denotes A different type of pseudoknot termed belaborated pseudoknotQ or bkissing stem loopsQ was found to be utilized by Human Coronavirus 229E (HCV 229E) (Herold and Siddell, 1993 ).
TextSentencer_T28 4177-4353 Sentence denotes A different type of pseudoknot termed belaborated pseudoknotQ or bkissing stem loopsQ was found to be utilized by Human Coronavirus 229E (HCV 229E) (Herold and Siddell, 1993 ).
TextSentencer_T29 4354-4468 Sentence denotes An alignment of frameshift cassettes from all available coronaviral sequences is shown in Fig. 1 , panels A and B.
TextSentencer_T29 4354-4468 Sentence denotes An alignment of frameshift cassettes from all available coronaviral sequences is shown in Fig. 1 , panels A and B.
TextSentencer_T30 4469-4564 Sentence denotes RNA secondary structures proposed based on this alignment are shown in Fig. 1 , panels C and D.
TextSentencer_T30 4469-4564 Sentence denotes RNA secondary structures proposed based on this alignment are shown in Fig. 1 , panels C and D.
TextSentencer_T31 4565-4705 Sentence denotes The alignment demonstrates the potential to utilize the belaborated pseudoknotQ only in the HCV 229E, HCV NL63, PEDV, and TGV coronaviruses.
TextSentencer_T31 4565-4705 Sentence denotes The alignment demonstrates the potential to utilize the belaborated pseudoknotQ only in the HCV 229E, HCV NL63, PEDV, and TGV coronaviruses.
TextSentencer_T32 4706-4843 Sentence denotes In the other coronaviruses, including SARS-CoV, the proposed structure is an RNA H-type pseudoknot similar to that characterized for IBV.
TextSentencer_T32 4706-4843 Sentence denotes In the other coronaviruses, including SARS-CoV, the proposed structure is an RNA H-type pseudoknot similar to that characterized for IBV.
TextSentencer_T33 4844-5057 Sentence denotes The predicted pseudoknot structure of SARS-CoV, the causative agent of Severe Acute Respiratory Syndrome (Marra et al., 2003; Thiel et al., 2003) , shares significant similarities to the IBV frameshift pseudoknot.
TextSentencer_T33 4844-5057 Sentence denotes The predicted pseudoknot structure of SARS-CoV, the causative agent of Severe Acute Respiratory Syndrome (Marra et al., 2003; Thiel et al., 2003) , shares significant similarities to the IBV frameshift pseudoknot.
TextSentencer_T34 5058-5204 Sentence denotes However, differences occur at sites likely to be important for frameshift stimulation, such as the junction between stem I and stem II (Fig. 1C) .
TextSentencer_T34 5058-5204 Sentence denotes However, differences occur at sites likely to be important for frameshift stimulation, such as the junction between stem I and stem II (Fig. 1C) .
TextSentencer_T35 5205-5473 Sentence denotes Recently, the existence of an RNA stem loop structure within the loop II of SARS-CoV pseudoknot has been proposed (Ramos et al., 2004) suggesting that unlike IBV there may be a critical feature in loop II of the SARS-CoV pseudoknot required for frameshift stimulation.
TextSentencer_T35 5205-5473 Sentence denotes Recently, the existence of an RNA stem loop structure within the loop II of SARS-CoV pseudoknot has been proposed (Ramos et al., 2004) suggesting that unlike IBV there may be a critical feature in loop II of the SARS-CoV pseudoknot required for frameshift stimulation.
TextSentencer_T36 5474-5708 Sentence denotes In addition to these differences in pseudoknot sequence and possibly structure, a second putative slippery site (G _ UUU _ UUA) partially overlaps the predicted standard heptanucleotide shift site, U _ UUA _ AAC (Fig. 1A) in SARS-CoV.
TextSentencer_T36 5474-5708 Sentence denotes In addition to these differences in pseudoknot sequence and possibly structure, a second putative slippery site (G _ UUU _ UUA) partially overlaps the predicted standard heptanucleotide shift site, U _ UUA _ AAC (Fig. 1A) in SARS-CoV.
TextSentencer_T37 5709-6004 Sentence denotes A similar slippery sequence (G _ UUA _ AAC) is used for frameshifting in Equine arteritis virus (Den Boon et al., 1991) indicating that the first position of the P-site tRNA anticodon (nucleotide 36) may not require traditional Watson Crick base pairing after shifting into the À1 reading frame.
TextSentencer_T37 5709-6004 Sentence denotes A similar slippery sequence (G _ UUA _ AAC) is used for frameshifting in Equine arteritis virus (Den Boon et al., 1991) indicating that the first position of the P-site tRNA anticodon (nucleotide 36) may not require traditional Watson Crick base pairing after shifting into the À1 reading frame.
TextSentencer_T38 6005-6155 Sentence denotes The present study is devoted to experimental analysis of the location, mechanism and the downstream stimulator of ribosomal frameshifting in SARS-CoV.
TextSentencer_T38 6005-6155 Sentence denotes The present study is devoted to experimental analysis of the location, mechanism and the downstream stimulator of ribosomal frameshifting in SARS-CoV.
TextSentencer_T39 6156-6280 Sentence denotes Comparisons of the frameshift regions from SARS-CoV and other coronaviruses demonstrate significant sequence homology (Figs.
TextSentencer_T39 6156-6280 Sentence denotes Comparisons of the frameshift regions from SARS-CoV and other coronaviruses demonstrate significant sequence homology (Figs.
TextSentencer_T40 6281-6292 Sentence denotes 1A and B) .
TextSentencer_T40 6281-6292 Sentence denotes 1A and B) .
TextSentencer_T41 6293-6418 Sentence denotes The potential for secondary structure is conserved due to compensatory variances that maintain base pairing as shown in Figs.
TextSentencer_T41 6293-6418 Sentence denotes The potential for secondary structure is conserved due to compensatory variances that maintain base pairing as shown in Figs.
TextSentencer_T42 6419-6428 Sentence denotes 1A and B.
TextSentencer_T42 6419-6428 Sentence denotes 1A and B.
TextSentencer_T43 6429-6571 Sentence denotes In particular, the secondary structure of stem I is preserved, with its distance from the frameshift site varying between 3 and 6 nucleotides.
TextSentencer_T43 6429-6571 Sentence denotes In particular, the secondary structure of stem I is preserved, with its distance from the frameshift site varying between 3 and 6 nucleotides.
TextSentencer_T44 6572-6588 Sentence denotes As seen in Figs.
TextSentencer_T44 6572-6588 Sentence denotes As seen in Figs.
TextSentencer_T45 6589-6688 Sentence denotes 1A and C, stem I varies between 11 and 14 base pairs, and stem II from 5 to 9 base pairs in length.
TextSentencer_T45 6589-6688 Sentence denotes 1A and C, stem I varies between 11 and 14 base pairs, and stem II from 5 to 9 base pairs in length.
TextSentencer_T46 6689-6847 Sentence denotes These values assume G:U and A:U pairs are formed at the ends of stems I and II although these pairings may be non-existent or only transiently formed in vivo.
TextSentencer_T46 6689-6847 Sentence denotes These values assume G:U and A:U pairs are formed at the ends of stems I and II although these pairings may be non-existent or only transiently formed in vivo.
TextSentencer_T47 6848-7025 Sentence denotes In three viruses, HCV 229E, HCV NL63, and Porcine Epidemic Diarrhea Virus (PEDV), there is a bulge inside the putative stem II that makes formation of this stem highly unlikely.
TextSentencer_T47 6848-7025 Sentence denotes In three viruses, HCV 229E, HCV NL63, and Porcine Epidemic Diarrhea Virus (PEDV), there is a bulge inside the putative stem II that makes formation of this stem highly unlikely.
TextSentencer_T48 7026-7169 Sentence denotes For HCV 229E, it has been shown that frameshifting is stimulated by a structure different from the one used in IBV (Herold and Siddell, 1993) .
TextSentencer_T48 7026-7169 Sentence denotes For HCV 229E, it has been shown that frameshifting is stimulated by a structure different from the one used in IBV (Herold and Siddell, 1993) .
TextSentencer_T49 7170-7336 Sentence denotes This involves pairing of the apical loop of stem I with the apical loop of stem III located downstream to form an delaborated pseudoknotT or kissing stem loops (Figs.
TextSentencer_T49 7170-7336 Sentence denotes This involves pairing of the apical loop of stem I with the apical loop of stem III located downstream to form an delaborated pseudoknotT or kissing stem loops (Figs.
TextSentencer_T50 7337-7344 Sentence denotes 1B, D).
TextSentencer_T50 7337-7344 Sentence denotes 1B, D).
TextSentencer_T51 7345-7433 Sentence denotes All coronaviral frameshift regions contain the shifty sequence U _ UUA _ AAC (Fig. 1A) .
TextSentencer_T51 7345-7433 Sentence denotes All coronaviral frameshift regions contain the shifty sequence U _ UUA _ AAC (Fig. 1A) .
TextSentencer_T52 7434-7521 Sentence denotes However, the SARS-CoV contains a second overlapping potential shift site G _ UUU _ UUA.
TextSentencer_T52 7434-7521 Sentence denotes However, the SARS-CoV contains a second overlapping potential shift site G _ UUU _ UUA.
TextSentencer_T53 7522-7700 Sentence denotes Frameshifting occurs in the Equine arteritis virus at the similar frameshift site, G _ UUA _ AAC (Den Boon et al., 1991) , where tRNALeu can pair with a GUU codon after slippage.
TextSentencer_T53 7522-7700 Sentence denotes Frameshifting occurs in the Equine arteritis virus at the similar frameshift site, G _ UUA _ AAC (Den Boon et al., 1991) , where tRNALeu can pair with a GUU codon after slippage.
TextSentencer_T54 7701-7927 Sentence denotes A distinctive feature of pseudoknots from SARS-CoV, MHV, BCV, and HCVOC43, which is not present in the well studied IBV pseudoknot, is the occurrence of two unpaired nucleotides at the stem I-stem II junction (Fig. 1A and C) .
TextSentencer_T54 7701-7927 Sentence denotes A distinctive feature of pseudoknots from SARS-CoV, MHV, BCV, and HCVOC43, which is not present in the well studied IBV pseudoknot, is the occurrence of two unpaired nucleotides at the stem I-stem II junction (Fig. 1A and C) .
TextSentencer_T55 7928-8055 Sentence denotes In addition, a second unique feature of SARS-CoV pseudoknot is the potential for two G:U pairs at the base of the stem I (Figs.
TextSentencer_T55 7928-8055 Sentence denotes In addition, a second unique feature of SARS-CoV pseudoknot is the potential for two G:U pairs at the base of the stem I (Figs.
TextSentencer_T56 8056-8066 Sentence denotes 1A and C).
TextSentencer_T56 8056-8066 Sentence denotes 1A and C).
TextSentencer_T57 8067-8154 Sentence denotes Comparative analysis does not provide a clear answer to whether these pairs are formed.
TextSentencer_T57 8067-8154 Sentence denotes Comparative analysis does not provide a clear answer to whether these pairs are formed.
TextSentencer_T58 8155-8280 Sentence denotes SARS-CoV frameshifting levels were measured in cultured cells utilizing a dual luciferase quantitative reporter assay system.
TextSentencer_T58 8155-8280 Sentence denotes SARS-CoV frameshifting levels were measured in cultured cells utilizing a dual luciferase quantitative reporter assay system.
TextSentencer_T59 8281-8512 Sentence denotes The reporter plasmid, containing the Renilla and firefly luciferase genes on either side of a multiple cloning site, can be expressed in transiently transfected tissue culture cells via the SV40 promoter (Grentzmann et al., 1998) .
TextSentencer_T59 8281-8512 Sentence denotes The reporter plasmid, containing the Renilla and firefly luciferase genes on either side of a multiple cloning site, can be expressed in transiently transfected tissue culture cells via the SV40 promoter (Grentzmann et al., 1998) .
TextSentencer_T60 8513-8656 Sentence denotes Sequences containing wild type and mutant variants of SARS-CoV frameshift site cassettes ( Fig. 2A) were cloned between the two reporter genes.
TextSentencer_T60 8513-8656 Sentence denotes Sequences containing wild type and mutant variants of SARS-CoV frameshift site cassettes ( Fig. 2A) were cloned between the two reporter genes.
TextSentencer_T61 8657-8809 Sentence denotes Constructs were designed such that the downstream firefly luciferase gene is in the À1 reading frame and can be translated only if frameshifting occurs.
TextSentencer_T61 8657-8809 Sentence denotes Constructs were designed such that the downstream firefly luciferase gene is in the À1 reading frame and can be translated only if frameshifting occurs.
TextSentencer_T62 8810-8973 Sentence denotes The dual luciferase reporter constructs were then transiently transfected into cultured HEK293 cells grown in 96-well plates as described in Materials and methods.
TextSentencer_T62 8810-8973 Sentence denotes The dual luciferase reporter constructs were then transiently transfected into cultured HEK293 cells grown in 96-well plates as described in Materials and methods.
TextSentencer_T63 8974-9110 Sentence denotes After 48 h of growth, cell lysates were prepared, and each luciferase activity was sequentially measured using an automated luminometer.
TextSentencer_T63 8974-9110 Sentence denotes After 48 h of growth, cell lysates were prepared, and each luciferase activity was sequentially measured using an automated luminometer.
TextSentencer_T64 9111-9277 Sentence denotes Efficiency of frameshifting was calculated as the difference in firefly luciferase activities normalized to Renilla luciferase expression (See Materials and methods).
TextSentencer_T64 9111-9277 Sentence denotes Efficiency of frameshifting was calculated as the difference in firefly luciferase activities normalized to Renilla luciferase expression (See Materials and methods).
TextSentencer_T65 9278-9357 Sentence denotes Values are shown as a percentage of the SARS-CoV wildtype frameshifting levels.
TextSentencer_T65 9278-9357 Sentence denotes Values are shown as a percentage of the SARS-CoV wildtype frameshifting levels.
TextSentencer_T66 9358-9672 Sentence denotes Frameshifting efficiencies are standardized to wildtype due to our observation that although relative changes in frameshifting levels remain consistent from experiment to experiment, the absolute frameshifting values can vary depending in part on the condition of the cultured cells (one factor is passage number).
TextSentencer_T66 9358-9672 Sentence denotes Frameshifting efficiencies are standardized to wildtype due to our observation that although relative changes in frameshifting levels remain consistent from experiment to experiment, the absolute frameshifting values can vary depending in part on the condition of the cultured cells (one factor is passage number).
TextSentencer_T67 9673-9843 Sentence denotes By standardizing frameshifting values to the absolute wildtype frameshifting levels observed in the same experiment, this experiment to experiment variation is corrected.
TextSentencer_T67 9673-9843 Sentence denotes By standardizing frameshifting values to the absolute wildtype frameshifting levels observed in the same experiment, this experiment to experiment variation is corrected.
TextSentencer_T68 9844-10015 Sentence denotes Absolute values for frameshifting efficiency at the wild type SARS-CoV and IBV recoding sites across all experiments was 15 F 3% and 12 F 3% in HEK293 cells, respectively.
TextSentencer_T68 9844-10015 Sentence denotes Absolute values for frameshifting efficiency at the wild type SARS-CoV and IBV recoding sites across all experiments was 15 F 3% and 12 F 3% in HEK293 cells, respectively.
TextSentencer_T69 10016-10253 Sentence denotes Changes of the codons corresponding to the P and A sites (U _ UUA _ AAC sequence to U _ UCA _ AAC or U _ UUA _ CAC) reduced frameshifting to background levels ( Fig. 2A ; FS1, FS2) consistent with a previous report (Thiel et al., 2003) .
TextSentencer_T69 10016-10253 Sentence denotes Changes of the codons corresponding to the P and A sites (U _ UUA _ AAC sequence to U _ UCA _ AAC or U _ UUA _ CAC) reduced frameshifting to background levels ( Fig. 2A ; FS1, FS2) consistent with a previous report (Thiel et al., 2003) .
TextSentencer_T70 10254-10400 Sentence denotes The difference between IBV and SARS-CoV sequences 5V of canonical shift site suggests that an alternative overlapping frameshift site may be used.
TextSentencer_T70 10254-10400 Sentence denotes The difference between IBV and SARS-CoV sequences 5V of canonical shift site suggests that an alternative overlapping frameshift site may be used.
TextSentencer_T71 10401-10591 Sentence denotes In SARS-CoV, the first four nucleotides of the U _ UUA _ AAC heptanucleotide shift site overlaps with a putative shift site G _ UUU _ UUA on which tandem slippage is hypothetically possible.
TextSentencer_T71 10401-10591 Sentence denotes In SARS-CoV, the first four nucleotides of the U _ UUA _ AAC heptanucleotide shift site overlaps with a putative shift site G _ UUU _ UUA on which tandem slippage is hypothetically possible.
TextSentencer_T72 10592-10758 Sentence denotes To check whether frameshifting occurs on this second shift site, several mutations of the U _ UUA _ AAC heptanucleotide sequence and 5Vflanking sequences were tested.
TextSentencer_T72 10592-10758 Sentence denotes To check whether frameshifting occurs on this second shift site, several mutations of the U _ UUA _ AAC heptanucleotide sequence and 5Vflanking sequences were tested.
TextSentencer_T73 10759-10996 Sentence denotes Changes of the two nucleotides upstream of the standard heptanucleotide motif did not reduce frameshifting efficiency ( Fig. 2B ; FS3-FS6), demonstrating that the G _ UUU _ UUA sequence does not contribute significantly to frameshifting.
TextSentencer_T73 10759-10996 Sentence denotes Changes of the two nucleotides upstream of the standard heptanucleotide motif did not reduce frameshifting efficiency ( Fig. 2B ; FS3-FS6), demonstrating that the G _ UUU _ UUA sequence does not contribute significantly to frameshifting.
TextSentencer_T74 10997-11187 Sentence denotes In fact, changing the nucleotide located 5V of the heptanucleotide shift site from a U to an A increases frameshifting efficiency by 1.6-fold relative to the wildtype levels ( Fig. 2; FS3 ).
TextSentencer_T74 10997-11187 Sentence denotes In fact, changing the nucleotide located 5V of the heptanucleotide shift site from a U to an A increases frameshifting efficiency by 1.6-fold relative to the wildtype levels ( Fig. 2; FS3 ).
TextSentencer_T75 11188-11369 Sentence denotes Bertrand et al. (2002) reported that the identity of the adjacent 3Vnucleotide can significantly impact frameshifting efficiency on the A _ AAA _ AAG shift site in Escherichia coli.
TextSentencer_T75 11188-11369 Sentence denotes Bertrand et al. (2002) reported that the identity of the adjacent 3Vnucleotide can significantly impact frameshifting efficiency on the A _ AAA _ AAG shift site in Escherichia coli.
TextSentencer_T76 11370-11546 Sentence denotes In addition, nucleotides within the spacer region have potential to form base pairs with strand I of stem II or nucleotides in strand II of stem II and adjacent 3V nucleotides.
TextSentencer_T76 11370-11546 Sentence denotes In addition, nucleotides within the spacer region have potential to form base pairs with strand I of stem II or nucleotides in strand II of stem II and adjacent 3V nucleotides.
TextSentencer_T77 11547-11613 Sentence denotes Either interaction may disrupt the predicted pseudoknot structure.
TextSentencer_T77 11547-11613 Sentence denotes Either interaction may disrupt the predicted pseudoknot structure.
TextSentencer_T78 11614-11894 Sentence denotes Interrupting this potential interaction by changing the three nucleotides downstream of the shift site in SARS-CoV had no significant effect on frameshifting levels ( Fig. 2A ; FS7 and FS8), suggesting that the identity of spacer region is not critical for frameshift stimulation.
TextSentencer_T78 11614-11894 Sentence denotes Interrupting this potential interaction by changing the three nucleotides downstream of the shift site in SARS-CoV had no significant effect on frameshifting levels ( Fig. 2A ; FS7 and FS8), suggesting that the identity of spacer region is not critical for frameshift stimulation.
TextSentencer_T79 11895-12131 Sentence denotes Experimental data reported for the IBV pseudoknot (Brierley et al., 1989) suggests that sequences forming the pseudoknot can be varied at some positions without loss of function if the secondary structure of the pseudoknot is preserved.
TextSentencer_T79 11895-12131 Sentence denotes Experimental data reported for the IBV pseudoknot (Brierley et al., 1989) suggests that sequences forming the pseudoknot can be varied at some positions without loss of function if the secondary structure of the pseudoknot is preserved.
TextSentencer_T80 12132-12271 Sentence denotes Mutagenic analysis of the SARS-CoV stem I and stem II was undertaken to determine critical features for ribosomal frameshifting (Fig. 3A) .
TextSentencer_T80 12132-12271 Sentence denotes Mutagenic analysis of the SARS-CoV stem I and stem II was undertaken to determine critical features for ribosomal frameshifting (Fig. 3A) .
TextSentencer_T81 12272-12514 Sentence denotes Disruption of stem I or II independently reduced frameshifting efficiency 10-to 20fold ( Fig. 3B ; SARSPK1, 3, and 5), equivalent to levels at which ribosomal frameshifting occurs at the À1 slippery sequence in the absence of any stimulators.
TextSentencer_T81 12272-12514 Sentence denotes Disruption of stem I or II independently reduced frameshifting efficiency 10-to 20fold ( Fig. 3B ; SARSPK1, 3, and 5), equivalent to levels at which ribosomal frameshifting occurs at the À1 slippery sequence in the absence of any stimulators.
TextSentencer_T82 12515-12742 Sentence denotes Restoration of the pseudoknot by bstrand switchingQ, which converts G:C, G:U, and A:U pairing to C:G, U:G, and U:A pairing, respectively ( Fig. 3A ; SARSPK4), of stem I restored frameshift levels to 180% of wildtype (Fig. 3B ).
TextSentencer_T82 12515-12742 Sentence denotes Restoration of the pseudoknot by bstrand switchingQ, which converts G:C, G:U, and A:U pairing to C:G, U:G, and U:A pairing, respectively ( Fig. 3A ; SARSPK4), of stem I restored frameshift levels to 180% of wildtype (Fig. 3B ).
TextSentencer_T83 12743-12987 Sentence denotes Changing the 5V half of stem I in SARSPK4 to the complementary sequence ( Fig. 3A ; SARSPK4-1) reduced frameshifting levels to 40% of wildtype ( Fig. 3B) suggesting that sequence as well as the structure of this portion of stem I are important.
TextSentencer_T83 12743-12987 Sentence denotes Changing the 5V half of stem I in SARSPK4 to the complementary sequence ( Fig. 3A ; SARSPK4-1) reduced frameshifting levels to 40% of wildtype ( Fig. 3B) suggesting that sequence as well as the structure of this portion of stem I are important.
TextSentencer_T84 12988-13248 Sentence denotes In agreement with this observation, when stem I was restored by bstem inversionQ (SARSPK2), in which the order of the base pairs in the stem are reordered from top to bottom ( Fig. 3A ; SARSPK2), frameshifting levels remained near background levels (Fig. 3B) .
TextSentencer_T84 12988-13248 Sentence denotes In agreement with this observation, when stem I was restored by bstem inversionQ (SARSPK2), in which the order of the base pairs in the stem are reordered from top to bottom ( Fig. 3A ; SARSPK2), frameshifting levels remained near background levels (Fig. 3B) .
TextSentencer_T85 13249-13363 Sentence denotes A similar result was obtained by a combination of bstrand switchingQ and bstem inversionQ for SARSPK2-1 (Fig. 3) .
TextSentencer_T85 13249-13363 Sentence denotes A similar result was obtained by a combination of bstrand switchingQ and bstem inversionQ for SARSPK2-1 (Fig. 3) .
TextSentencer_T86 13364-13489 Sentence denotes Each altered sequence was examined using mfold (Mathews et al., 1999; Zuker, 2003) for other possible folding configurations.
TextSentencer_T86 13364-13489 Sentence denotes Each altered sequence was examined using mfold (Mathews et al., 1999; Zuker, 2003) for other possible folding configurations.
TextSentencer_T87 13490-13628 Sentence denotes In each case where base pairing potential was restored mfold predicted the expected pairing of strand I and II of stem I (data not shown).
TextSentencer_T87 13490-13628 Sentence denotes In each case where base pairing potential was restored mfold predicted the expected pairing of strand I and II of stem I (data not shown).
TextSentencer_T88 13629-13826 Sentence denotes Restoration of stem II (SARSPK6) by bstrand switchingQ returned frameshifting to wildtype levels. bStem II inver-sionQ resulted in a reduction of frameshifting SARSPK6A (Fig. 3) to 20% of wildtype.
TextSentencer_T88 13629-13826 Sentence denotes Restoration of stem II (SARSPK6) by bstrand switchingQ returned frameshifting to wildtype levels. bStem II inver-sionQ resulted in a reduction of frameshifting SARSPK6A (Fig. 3) to 20% of wildtype.
TextSentencer_T89 13827-14010 Sentence denotes Changing the three C:G pairs to A:U pairs (SARSPK7) further reduced frameshifting levels to background and bstem switchingQ of SARSPK7 (SARSPK7A) did not change this level (Fig. 3B) .
TextSentencer_T89 13827-14010 Sentence denotes Changing the three C:G pairs to A:U pairs (SARSPK7) further reduced frameshifting levels to background and bstem switchingQ of SARSPK7 (SARSPK7A) did not change this level (Fig. 3B) .
TextSentencer_T90 14011-14134 Sentence denotes As for stem I, it appears that both pairing potential and the sequence of stem II are important for frameshift stimulation.
TextSentencer_T90 14011-14134 Sentence denotes As for stem I, it appears that both pairing potential and the sequence of stem II are important for frameshift stimulation.
TextSentencer_T91 14135-14325 Sentence denotes According to experiments performed with the IBV pseudoknot, the sequence of loop II and its length can be varied significantly (Brierley et al., 1991) without altering frameshift efficiency.
TextSentencer_T91 14135-14325 Sentence denotes According to experiments performed with the IBV pseudoknot, the sequence of loop II and its length can be varied significantly (Brierley et al., 1991) without altering frameshift efficiency.
TextSentencer_T92 14326-14467 Sentence denotes For the SARS-CoV pseudoknot, it has recently been proposed that a stem loop structure can be formed within the loop II (Ramos et al., 2004) .
TextSentencer_T92 14326-14467 Sentence denotes For the SARS-CoV pseudoknot, it has recently been proposed that a stem loop structure can be formed within the loop II (Ramos et al., 2004) .
TextSentencer_T93 14468-14659 Sentence denotes To determine the importance of loop II in SARS-CoV, sequence portions of nine nucleotides at a time were deleted from the 3Vend of loop II and frameshift levels were measured in HEK293 cells.
TextSentencer_T93 14468-14659 Sentence denotes To determine the importance of loop II in SARS-CoV, sequence portions of nine nucleotides at a time were deleted from the 3Vend of loop II and frameshift levels were measured in HEK293 cells.
TextSentencer_T94 14660-14725 Sentence denotes The first deletion had no significant effect ( Fig. 4B ; SARSL1).
TextSentencer_T94 14660-14725 Sentence denotes The first deletion had no significant effect ( Fig. 4B ; SARSL1).
TextSentencer_T95 14726-14912 Sentence denotes Deleting 18 nucleotides reduced frameshifting efficiency slightly to 80% ( Fig. 4B ; SARSL2) and further reduced to 35% of wildtype levels by deleting 27 nucleotides ( Fig. 4B; SARSL3) .
TextSentencer_T95 14726-14912 Sentence denotes Deleting 18 nucleotides reduced frameshifting efficiency slightly to 80% ( Fig. 4B ; SARSL2) and further reduced to 35% of wildtype levels by deleting 27 nucleotides ( Fig. 4B; SARSL3) .
TextSentencer_T96 14913-15047 Sentence denotes Surprisingly, a dramatic reduction in frameshifting was observed by leaving the length unchanged but altering the sequence of loop II.
TextSentencer_T96 14913-15047 Sentence denotes Surprisingly, a dramatic reduction in frameshifting was observed by leaving the length unchanged but altering the sequence of loop II.
TextSentencer_T97 15048-15118 Sentence denotes Nine nucleotides at a time were changed to the complimentary sequence.
TextSentencer_T97 15048-15118 Sentence denotes Nine nucleotides at a time were changed to the complimentary sequence.
TextSentencer_T98 15119-15250 Sentence denotes Frameshifting was reduced to 35% of wildtype when 9 nucleotides located near the 3V end of loop II were changed ( Fig. 4; SARSL4 ).
TextSentencer_T98 15119-15250 Sentence denotes Frameshifting was reduced to 35% of wildtype when 9 nucleotides located near the 3V end of loop II were changed ( Fig. 4; SARSL4 ).
TextSentencer_T99 15251-15440 Sentence denotes Most strikingly, changes in the middle or near the 5Vend of the loop reduced frameshifting levels to 7% and 8% of wildtype frameshifting levels, respectively ( Fig. 4 ; SARSL5, and SARSL6).
TextSentencer_T99 15251-15440 Sentence denotes Most strikingly, changes in the middle or near the 5Vend of the loop reduced frameshifting levels to 7% and 8% of wildtype frameshifting levels, respectively ( Fig. 4 ; SARSL5, and SARSL6).
TextSentencer_T100 15441-15523 Sentence denotes There is a potential for RNA stem loop formation in loop II (Ramos et al., 2004) .
TextSentencer_T100 15441-15523 Sentence denotes There is a potential for RNA stem loop formation in loop II (Ramos et al., 2004) .
TextSentencer_T101 15524-15729 Sentence denotes Its importance for the frameshifting is not supported by the deletion mutants, in SARSL1 and SARSL2 in which a significant part of this structure is removed (Fig. 4) , but frameshifting levels remain high.
TextSentencer_T101 15524-15729 Sentence denotes Its importance for the frameshifting is not supported by the deletion mutants, in SARSL1 and SARSL2 in which a significant part of this structure is removed (Fig. 4) , but frameshifting levels remain high.
TextSentencer_T102 15730-15832 Sentence denotes However, it is possible that this stem loop is important in the context of the wildtype large loop II.
TextSentencer_T102 15730-15832 Sentence denotes However, it is possible that this stem loop is important in the context of the wildtype large loop II.
TextSentencer_T103 15833-16058 Sentence denotes To test this, we restored formation of the RNA stem loop in loop II by converting G:C and A:U pairs to C:G and U:A pairs respectively, but this change did not result in restoration of frameshifting levels ( Fig. 4B; SARSL7) .
TextSentencer_T103 15833-16058 Sentence denotes To test this, we restored formation of the RNA stem loop in loop II by converting G:C and A:U pairs to C:G and U:A pairs respectively, but this change did not result in restoration of frameshifting levels ( Fig. 4B; SARSL7) .
TextSentencer_T104 16059-16130 Sentence denotes A notable feature of the WT stem loop is the presence of two bulged As.
TextSentencer_T104 16059-16130 Sentence denotes A notable feature of the WT stem loop is the presence of two bulged As.
TextSentencer_T105 16131-16280 Sentence denotes Unpaired or bulged adenosines are known to form A-minor motifs, a very common element of tertiary structure interactions (reviewed in Strobel 2002) .
TextSentencer_T105 16131-16280 Sentence denotes Unpaired or bulged adenosines are known to form A-minor motifs, a very common element of tertiary structure interactions (reviewed in Strobel 2002) .
TextSentencer_T106 16281-16381 Sentence denotes In the SARSL7 construct, the bulged nucleotides in the predicted stems are changed to either C or U.
TextSentencer_T106 16281-16381 Sentence denotes In the SARSL7 construct, the bulged nucleotides in the predicted stems are changed to either C or U.
TextSentencer_T107 16382-16532 Sentence denotes By changing these nucleotides and the 3V U in the apical loop to As ( Fig. 4; SARSL8) , frameshifting levels were restored to nearly wild type levels.
TextSentencer_T107 16382-16532 Sentence denotes By changing these nucleotides and the 3V U in the apical loop to As ( Fig. 4; SARSL8) , frameshifting levels were restored to nearly wild type levels.
TextSentencer_T108 16533-16734 Sentence denotes To further confirm the importance of this stem loop, we made minimal changes (only three nucleotides were mutated) which should significantly disturb the structure of the stem loop ( Fig. 4; SARSPK9) .
TextSentencer_T108 16533-16734 Sentence denotes To further confirm the importance of this stem loop, we made minimal changes (only three nucleotides were mutated) which should significantly disturb the structure of the stem loop ( Fig. 4; SARSPK9) .
TextSentencer_T109 16735-16800 Sentence denotes These minimal changes reduced frameshifting to background levels.
TextSentencer_T109 16735-16800 Sentence denotes These minimal changes reduced frameshifting to background levels.
TextSentencer_T110 16801-16962 Sentence denotes One difference noted in sequence comparisons between IBV and SARS-CoV pseudoknots was the presence of unpaired GU nucleotides near the stem junction in SARS-CoV.
TextSentencer_T110 16801-16962 Sentence denotes One difference noted in sequence comparisons between IBV and SARS-CoV pseudoknots was the presence of unpaired GU nucleotides near the stem junction in SARS-CoV.
TextSentencer_T111 16963-17080 Sentence denotes SARSLTG and SARSLAC were constructed to test the importance of this dinucleotide to frameshift stimulation (Fig. 4) .
TextSentencer_T111 16963-17080 Sentence denotes SARSLTG and SARSLAC were constructed to test the importance of this dinucleotide to frameshift stimulation (Fig. 4) .
TextSentencer_T112 17081-17225 Sentence denotes SARSLTG extends the base pairing potential of stem I by creating complementary nucleotides between stem I and the 5V most nucleotides of loop I.
TextSentencer_T112 17081-17225 Sentence denotes SARSLTG extends the base pairing potential of stem I by creating complementary nucleotides between stem I and the 5V most nucleotides of loop I.
TextSentencer_T113 17226-17347 Sentence denotes In SARSLAC, the unpaired GU nucleotides are changed to AC which maintains these nucleotides in an unpaired configuration.
TextSentencer_T113 17226-17347 Sentence denotes In SARSLAC, the unpaired GU nucleotides are changed to AC which maintains these nucleotides in an unpaired configuration.
TextSentencer_T114 17348-17517 Sentence denotes The predicted secondary structure of SARSLTG pseudoknot is similar to the IBV secondary structure where this extended pairing potential in stem I is observed (Fig. 1B) .
TextSentencer_T114 17348-17517 Sentence denotes The predicted secondary structure of SARSLTG pseudoknot is similar to the IBV secondary structure where this extended pairing potential in stem I is observed (Fig. 1B) .
TextSentencer_T115 17518-17719 Sentence denotes Both alterations reduced the ability of this pseudoknot to stimulate frameshifting to 15% of wildtype levels (Fig. 4B ) demonstrating the importance of these two nucleotides for frameshift stimulation.
TextSentencer_T115 17518-17719 Sentence denotes Both alterations reduced the ability of this pseudoknot to stimulate frameshifting to 15% of wildtype levels (Fig. 4B ) demonstrating the importance of these two nucleotides for frameshift stimulation.
TextSentencer_T116 17720-17935 Sentence denotes Frameshifting for gene expression purposes in viral genes frequently occurs on a heptanucleotide bslipperyQ motif, X _ XXY _ YYZ (Baranov et al., 2002; Hatfield et al., 1992; Jacks et al., 1988; Namy et al., 2004) .
TextSentencer_T116 17720-17935 Sentence denotes Frameshifting for gene expression purposes in viral genes frequently occurs on a heptanucleotide bslipperyQ motif, X _ XXY _ YYZ (Baranov et al., 2002; Hatfield et al., 1992; Jacks et al., 1988; Namy et al., 2004) .
TextSentencer_T117 17936-18080 Sentence denotes A common assumption is that P-and A-site tRNAs decoding XXY and YYZ, reposition into the À1 reading frame at the overlapping XXX and YYY codons.
TextSentencer_T117 17936-18080 Sentence denotes A common assumption is that P-and A-site tRNAs decoding XXY and YYZ, reposition into the À1 reading frame at the overlapping XXX and YYY codons.
TextSentencer_T118 18081-18286 Sentence denotes However, this is not always the case as frameshifting in decoding an HIV frameshift cassette results in production of two protein products when it is expressed in reticulocyte lysate (Jacks et al., 1988) .
TextSentencer_T118 18081-18286 Sentence denotes However, this is not always the case as frameshifting in decoding an HIV frameshift cassette results in production of two protein products when it is expressed in reticulocyte lysate (Jacks et al., 1988) .
TextSentencer_T119 18287-18624 Sentence denotes While 70% of the product corresponds to a product of tandem slippage of A-and P-site tRNAs, there is an additional product corresponding to frameshifting that involves either slippage of E-and P-site tRNAs (Horsfield et al., 1995) or other tRNA rearrangements inside the ribosome allowing À1 P-site tRNA slippage (Baranov et al., 2004) .
TextSentencer_T119 18287-18624 Sentence denotes While 70% of the product corresponds to a product of tandem slippage of A-and P-site tRNAs, there is an additional product corresponding to frameshifting that involves either slippage of E-and P-site tRNAs (Horsfield et al., 1995) or other tRNA rearrangements inside the ribosome allowing À1 P-site tRNA slippage (Baranov et al., 2004) .
TextSentencer_T120 18625-18769 Sentence denotes To examine whether a similar situation occurs during frameshifting in SARS-CoV, a molecular mass of the frameshifting product(s) was determined.
TextSentencer_T120 18625-18769 Sentence denotes To examine whether a similar situation occurs during frameshifting in SARS-CoV, a molecular mass of the frameshifting product(s) was determined.
TextSentencer_T121 18770-18976 Sentence denotes The SARS-CoV frameshift region was cloned into a mammalian expression vector, flanked upstream by Glutathione-S transferase and downstream by a six histidine nickel affinity tag (see Materials and methods).
TextSentencer_T121 18770-18976 Sentence denotes The SARS-CoV frameshift region was cloned into a mammalian expression vector, flanked upstream by Glutathione-S transferase and downstream by a six histidine nickel affinity tag (see Materials and methods).
TextSentencer_T122 18977-19114 Sentence denotes This recombinant construct was expressed in cultured cells and the products of its expression analyzed by electrospray mass spectrometry.
TextSentencer_T122 18977-19114 Sentence denotes This recombinant construct was expressed in cultured cells and the products of its expression analyzed by electrospray mass spectrometry.
TextSentencer_T123 19115-19325 Sentence denotes Only one significant product was detected ( Fig. 5 ; observed MW = 45703), corresponding to the predicted mass (MW = 45703) of a protein product with leucine and asparagine being incorporated at the shift site.
TextSentencer_T123 19115-19325 Sentence denotes Only one significant product was detected ( Fig. 5 ; observed MW = 45703), corresponding to the predicted mass (MW = 45703) of a protein product with leucine and asparagine being incorporated at the shift site.
TextSentencer_T124 19326-19525 Sentence denotes This result clearly indicates that frameshifting in SARS-CoV occurs only due to tandem slippage of the P-site tRNALeu and A-site tRNAAsn which takes place at the standard U _ UUA _ AAC slippery site.
TextSentencer_T124 19326-19525 Sentence denotes This result clearly indicates that frameshifting in SARS-CoV occurs only due to tandem slippage of the P-site tRNALeu and A-site tRNAAsn which takes place at the standard U _ UUA _ AAC slippery site.
TextSentencer_T125 19526-19839 Sentence denotes Although 1 tandem tRNA slippage is often assumed to be the mechanism ribosome frameshifting at X _ XXY _ YYZ sequence motifs, À1 P-site slippage is possible at these motifs and this alternative mechanism has been shown to account for approximately 30% of frameshifting at the HIV shift site (Jacks et al., 1988) .
TextSentencer_T125 19526-19839 Sentence denotes Although 1 tandem tRNA slippage is often assumed to be the mechanism ribosome frameshifting at X _ XXY _ YYZ sequence motifs, À1 P-site slippage is possible at these motifs and this alternative mechanism has been shown to account for approximately 30% of frameshifting at the HIV shift site (Jacks et al., 1988) .
TextSentencer_T126 19840-20128 Sentence denotes Mutagenic analysis of the SARS-CoV heptanucleotide frameshift site presented here, suggests that frameshifting occurs at the predicted site and most probably by a tandem tRNA slippage mechanism as frameshifting levels are significantly reduced by changes in either the A-or P-site codons.
TextSentencer_T126 19840-20128 Sentence denotes Mutagenic analysis of the SARS-CoV heptanucleotide frameshift site presented here, suggests that frameshifting occurs at the predicted site and most probably by a tandem tRNA slippage mechanism as frameshifting levels are significantly reduced by changes in either the A-or P-site codons.
TextSentencer_T127 20129-20306 Sentence denotes To confirm the mechanism of frameshifting at this site, we report mass spectrometry of the protein product arising from frameshifting on the coronaviral frameshift site in vivo.
TextSentencer_T127 20129-20306 Sentence denotes To confirm the mechanism of frameshifting at this site, we report mass spectrometry of the protein product arising from frameshifting on the coronaviral frameshift site in vivo.
TextSentencer_T128 20307-20447 Sentence denotes The resulting mass indicates that frameshifting occurs after 0-frame decoding of the Asn codon and a single trans-frame product is produced.
TextSentencer_T128 20307-20447 Sentence denotes The resulting mass indicates that frameshifting occurs after 0-frame decoding of the Asn codon and a single trans-frame product is produced.
TextSentencer_T129 20448-20604 Sentence denotes This result when combined with mutagenic analysis of the SARS-CoV shift site definitively identifies the mechanism of frameshifting as tandem tRNA slippage.
TextSentencer_T129 20448-20604 Sentence denotes This result when combined with mutagenic analysis of the SARS-CoV shift site definitively identifies the mechanism of frameshifting as tandem tRNA slippage.
TextSentencer_T130 20605-20759 Sentence denotes This shows the utility of mass spectrometry analysis for determination of the location of recoding events, and for elucidation of the mechanisms involved.
TextSentencer_T130 20605-20759 Sentence denotes This shows the utility of mass spectrometry analysis for determination of the location of recoding events, and for elucidation of the mechanisms involved.
TextSentencer_T131 20760-20844 Sentence denotes Frameshifting efficiency at the wild type IBV recoding site was 12% in HEK293 cells.
TextSentencer_T131 20760-20844 Sentence denotes Frameshifting efficiency at the wild type IBV recoding site was 12% in HEK293 cells.
TextSentencer_T132 20845-20987 Sentence denotes IBV frameshifting efficiency is lower than what has previously been reported using rabbit reticulocyte lysates (30%) (Brierley et al., 1989) .
TextSentencer_T132 20845-20987 Sentence denotes IBV frameshifting efficiency is lower than what has previously been reported using rabbit reticulocyte lysates (30%) (Brierley et al., 1989) .
TextSentencer_T133 20988-21262 Sentence denotes However, this is not surprising since discrepancies in absolute values of frameshifting measured in different systems are not uncommon and it was observed that, in general, frameshifting efficiency is lower when measured in vivo (Grentzmann et al., 1998; Kim et al., 1999) .
TextSentencer_T133 20988-21262 Sentence denotes However, this is not surprising since discrepancies in absolute values of frameshifting measured in different systems are not uncommon and it was observed that, in general, frameshifting efficiency is lower when measured in vivo (Grentzmann et al., 1998; Kim et al., 1999) .
TextSentencer_T134 21263-21348 Sentence denotes The critical difference between in vitro and in vivo assays remains to be identified.
TextSentencer_T134 21263-21348 Sentence denotes The critical difference between in vitro and in vivo assays remains to be identified.
TextSentencer_T135 21349-21530 Sentence denotes A recent report of IBV frameshifting using the dual luciferase reporter system in cultured cells found IBV frameshifting to be even lower at approximately 4% (Ivanov et al., 2004) .
TextSentencer_T135 21349-21530 Sentence denotes A recent report of IBV frameshifting using the dual luciferase reporter system in cultured cells found IBV frameshifting to be even lower at approximately 4% (Ivanov et al., 2004) .
TextSentencer_T136 21531-21775 Sentence denotes The lower level frameshifting reported in that study was found to be due to the exclusion of several nucleotides just downstream from the 3V end of the IBV pseudoknot which are required for the higher level frameshifting observed in this study.
TextSentencer_T136 21531-21775 Sentence denotes The lower level frameshifting reported in that study was found to be due to the exclusion of several nucleotides just downstream from the 3V end of the IBV pseudoknot which are required for the higher level frameshifting observed in this study.
TextSentencer_T137 21776-22009 Sentence denotes Nevertheless, comparable frameshifting efficiencies were observed for the IBV and SARS-CoV reporter constructs containing the larger frameshift window used in the present study (see Materials and methods for the IBV sequence tested).
TextSentencer_T137 21776-22009 Sentence denotes Nevertheless, comparable frameshifting efficiencies were observed for the IBV and SARS-CoV reporter constructs containing the larger frameshift window used in the present study (see Materials and methods for the IBV sequence tested).
TextSentencer_T138 22010-22113 Sentence denotes Site-directed mutagenesis of the heptanucleotide motif reduced frameshifting levels to near background.
TextSentencer_T138 22010-22113 Sentence denotes Site-directed mutagenesis of the heptanucleotide motif reduced frameshifting levels to near background.
TextSentencer_T139 22114-22347 Sentence denotes Whereas, changing the immediate surrounding sequence had no significant effect on frameshifting with the exception that altering the nucleotide preceding the heptanucleotide motif to an A produced an increase in frameshifting levels.
TextSentencer_T139 22114-22347 Sentence denotes Whereas, changing the immediate surrounding sequence had no significant effect on frameshifting with the exception that altering the nucleotide preceding the heptanucleotide motif to an A produced an increase in frameshifting levels.
TextSentencer_T140 22348-22485 Sentence denotes Interestingly, in approximately half of the other coronaviruses with known genomic sequences, an A occurs in this position (See Fig. 1) .
TextSentencer_T140 22348-22485 Sentence denotes Interestingly, in approximately half of the other coronaviruses with known genomic sequences, an A occurs in this position (See Fig. 1) .
TextSentencer_T141 22486-22630 Sentence denotes A possibility for increased frameshifting in this case could be the change of encoded amino acid and consequent effect from the nascent peptide.
TextSentencer_T141 22486-22630 Sentence denotes A possibility for increased frameshifting in this case could be the change of encoded amino acid and consequent effect from the nascent peptide.
TextSentencer_T142 22631-22717 Sentence denotes However, other mutations leading to amino acid changes do not produce the same effect.
TextSentencer_T142 22631-22717 Sentence denotes However, other mutations leading to amino acid changes do not produce the same effect.
TextSentencer_T143 22718-22902 Sentence denotes More likely, this effect is achieved through interaction between the nucleotide preceding and the first nucleotide of the heptanucleotide motif which affects the P-site tRNA stability.
TextSentencer_T143 22718-22902 Sentence denotes More likely, this effect is achieved through interaction between the nucleotide preceding and the first nucleotide of the heptanucleotide motif which affects the P-site tRNA stability.
TextSentencer_T144 22903-23117 Sentence denotes Alternatively, this effect can be achieved through interactions with the E-site tRNA, as it was recently proposed as a general factor influencing À1 frameshifting in eukaryotic viruses (Bekaert and Rousset, 2005) .
TextSentencer_T144 22903-23117 Sentence denotes Alternatively, this effect can be achieved through interactions with the E-site tRNA, as it was recently proposed as a general factor influencing À1 frameshifting in eukaryotic viruses (Bekaert and Rousset, 2005) .
TextSentencer_T145 23118-23327 Sentence denotes The importance of base pairing potential of the sequences forming stem I of the SARS-CoV pseudoknot is demonstrated by alterations which eliminate this potential or reverse the order of base pairing (Fig. 3) .
TextSentencer_T145 23118-23327 Sentence denotes The importance of base pairing potential of the sequences forming stem I of the SARS-CoV pseudoknot is demonstrated by alterations which eliminate this potential or reverse the order of base pairing (Fig. 3) .
TextSentencer_T146 23328-23462 Sentence denotes However, when the sequence of stem I was reversed from top to bottom but base pairing maintained, frameshifting was severely impaired.
TextSentencer_T146 23328-23462 Sentence denotes However, when the sequence of stem I was reversed from top to bottom but base pairing maintained, frameshifting was severely impaired.
TextSentencer_T147 23463-23514 Sentence denotes At least three factors may account for this effect.
TextSentencer_T147 23463-23514 Sentence denotes At least three factors may account for this effect.
TextSentencer_T148 23515-23572 Sentence denotes Stem I is shown on Fig. 1 with two G:U pairs at its base.
TextSentencer_T148 23515-23572 Sentence denotes Stem I is shown on Fig. 1 with two G:U pairs at its base.
TextSentencer_T149 23573-23623 Sentence denotes It is unclear whether these base pairs are formed.
TextSentencer_T149 23573-23623 Sentence denotes It is unclear whether these base pairs are formed.
TextSentencer_T150 23624-23749 Sentence denotes If the spacer between the pseudoknot and frameshift site is the same in IBV and SARS-CoV, only one G:U pair should be formed.
TextSentencer_T150 23624-23749 Sentence denotes If the spacer between the pseudoknot and frameshift site is the same in IBV and SARS-CoV, only one G:U pair should be formed.
TextSentencer_T151 23750-23911 Sentence denotes Reversal of stem I from top to bottom (Fig. 3, SARSPK2) potentially changes either the spacer between the frameshift site and pseudoknot or the length of stem I.
TextSentencer_T151 23750-23911 Sentence denotes Reversal of stem I from top to bottom (Fig. 3, SARSPK2) potentially changes either the spacer between the frameshift site and pseudoknot or the length of stem I.
TextSentencer_T152 23912-24142 Sentence denotes Spacer length seems unlikely to be responsible for the loss of activity as small changes in spacing do not eliminate frameshifting in IBV but reduce efficiency by only several fold (Brierley et al., 1989 (Brierley et al., , 1992 .
TextSentencer_T152 23912-24142 Sentence denotes Spacer length seems unlikely to be responsible for the loss of activity as small changes in spacing do not eliminate frameshifting in IBV but reduce efficiency by only several fold (Brierley et al., 1989 (Brierley et al., , 1992 .
TextSentencer_T153 24143-24239 Sentence denotes Another consideration is that stem I in SARS-CoV is more G:C rich at the bottom than at the top.
TextSentencer_T153 24143-24239 Sentence denotes Another consideration is that stem I in SARS-CoV is more G:C rich at the bottom than at the top.
TextSentencer_T154 24240-24311 Sentence denotes The majority of viral pseudoknots are G:C rich at the bottom of stem I.
TextSentencer_T154 24240-24311 Sentence denotes The majority of viral pseudoknots are G:C rich at the bottom of stem I.
TextSentencer_T155 24312-24469 Sentence denotes The importance of this feature was demonstrated for IBV previously where changes of G:C pairs in the stem I to A:U pairs reduced frameshifting about 6-fold .
TextSentencer_T155 24312-24469 Sentence denotes The importance of this feature was demonstrated for IBV previously where changes of G:C pairs in the stem I to A:U pairs reduced frameshifting about 6-fold .
TextSentencer_T156 24470-24608 Sentence denotes The stability of this region of frameshift stimulators is likely to be a generally important feature for inducing ribosomal frameshifting.
TextSentencer_T156 24470-24608 Sentence denotes The stability of this region of frameshift stimulators is likely to be a generally important feature for inducing ribosomal frameshifting.
TextSentencer_T157 24609-24706 Sentence denotes Most À1 frameshift stimulators start 5-9 nt downstream from the end of the heptanucleotide motif.
TextSentencer_T157 24609-24706 Sentence denotes Most À1 frameshift stimulators start 5-9 nt downstream from the end of the heptanucleotide motif.
TextSentencer_T158 24707-25005 Sentence denotes The recent crystal structure of the T. thermophilus ribosome with mRNA diffused into the crystal suggests that the mRNA begins to enter the ribosome 7-9 nt downstream from P-site (Yusupova et al., 2001 ) (placing these À1 stimulatory structures very near the entrance to the ribosome mRNA channel).
TextSentencer_T158 24707-25005 Sentence denotes The recent crystal structure of the T. thermophilus ribosome with mRNA diffused into the crystal suggests that the mRNA begins to enter the ribosome 7-9 nt downstream from P-site (Yusupova et al., 2001 ) (placing these À1 stimulatory structures very near the entrance to the ribosome mRNA channel).
TextSentencer_T159 25006-25154 Sentence denotes During elongation, ribosomes must unwind the stem starting from this G:C rich region while the P-and A-sites are decoding the heptanucleotide motif.
TextSentencer_T159 25006-25154 Sentence denotes During elongation, ribosomes must unwind the stem starting from this G:C rich region while the P-and A-sites are decoding the heptanucleotide motif.
TextSentencer_T160 25155-25293 Sentence denotes This observation provides a rationale for the importance of stability in this part of the pseudoknot and its distance from the shift site.
TextSentencer_T160 25155-25293 Sentence denotes This observation provides a rationale for the importance of stability in this part of the pseudoknot and its distance from the shift site.
TextSentencer_T161 25294-25484 Sentence denotes Deletions in the IBV loop (data not shown) corresponding to SARSL1 and SARSL2 did not reduce frameshifting, whereas an IBV deletion corresponding to SARSL3 decreased frameshifting by 4-fold.
TextSentencer_T161 25294-25484 Sentence denotes Deletions in the IBV loop (data not shown) corresponding to SARSL1 and SARSL2 did not reduce frameshifting, whereas an IBV deletion corresponding to SARSL3 decreased frameshifting by 4-fold.
TextSentencer_T162 25485-25670 Sentence denotes The large deletion in SARSL3 and the corresponding deletion in IBV reduce the loop size to 5 nt which may be too short to span the distance from stems I and II (Brierley et al., 1991) .
TextSentencer_T162 25485-25670 Sentence denotes The large deletion in SARSL3 and the corresponding deletion in IBV reduce the loop size to 5 nt which may be too short to span the distance from stems I and II (Brierley et al., 1991) .
TextSentencer_T163 25671-25781 Sentence denotes More markedly, changing the sequence of the loop II in SARS-CoV dramatically reduced frameshifting efficiency.
TextSentencer_T163 25671-25781 Sentence denotes More markedly, changing the sequence of the loop II in SARS-CoV dramatically reduced frameshifting efficiency.
TextSentencer_T164 25782-25893 Sentence denotes This suggests that loop II may contribute to important structural features required for frameshift stimulation.
TextSentencer_T164 25782-25893 Sentence denotes This suggests that loop II may contribute to important structural features required for frameshift stimulation.
TextSentencer_T165 25894-25992 Sentence denotes An additional stem loop structure within loop II has been proposed recently (Ramos et al., 2004) .
TextSentencer_T165 25894-25992 Sentence denotes An additional stem loop structure within loop II has been proposed recently (Ramos et al., 2004) .
TextSentencer_T166 25993-26167 Sentence denotes This stem loop is not required within loop II to support high efficiency of frameshifting, since deletions in the loop II do not significantly alter frameshifting efficiency.
TextSentencer_T166 25993-26167 Sentence denotes This stem loop is not required within loop II to support high efficiency of frameshifting, since deletions in the loop II do not significantly alter frameshifting efficiency.
TextSentencer_T167 26168-26285 Sentence denotes Evolutionary importance of this stem loop is not supported by comparative sequence analysis with other coronaviruses.
TextSentencer_T167 26168-26285 Sentence denotes Evolutionary importance of this stem loop is not supported by comparative sequence analysis with other coronaviruses.
TextSentencer_T168 26286-26408 Sentence denotes However, our data suggest that in the WT context of a large loop II, it plays an important role in frameshift stimulation.
TextSentencer_T168 26286-26408 Sentence denotes However, our data suggest that in the WT context of a large loop II, it plays an important role in frameshift stimulation.
TextSentencer_T169 26409-26559 Sentence denotes Most likely, the RNA in loop II (if not properly structured) can interfere with the structure of the stimulating pseudoknot and reduce its efficiency.
TextSentencer_T169 26409-26559 Sentence denotes Most likely, the RNA in loop II (if not properly structured) can interfere with the structure of the stimulating pseudoknot and reduce its efficiency.
TextSentencer_T170 26560-26832 Sentence denotes Our results demonstrate that the bulged As in this proposed stem loop are important for frameshifting efficiency, potentially through interactions with the rest of pseudoknot body (for example, by A-minor motif interactions (Battle and Doudna, 2002; Nissen et al., 2001) .
TextSentencer_T170 26560-26832 Sentence denotes Our results demonstrate that the bulged As in this proposed stem loop are important for frameshifting efficiency, potentially through interactions with the rest of pseudoknot body (for example, by A-minor motif interactions (Battle and Doudna, 2002; Nissen et al., 2001) .
TextSentencer_T171 26833-27089 Sentence denotes The importance of this stem loop for high efficiency frameshifting in SARS-CoV is further supported by the fact that substitutions of only three nucleotides important for the formation of this stem loop reduce frameshifting efficiency to background levels.
TextSentencer_T171 26833-27089 Sentence denotes The importance of this stem loop for high efficiency frameshifting in SARS-CoV is further supported by the fact that substitutions of only three nucleotides important for the formation of this stem loop reduce frameshifting efficiency to background levels.
TextSentencer_T172 27090-27322 Sentence denotes An answer to whether loop IIs in other coronoviruses need proper folding and to what extent they can accommodate sequence changes without loss of frameshifting requires more extensive studies of pseudoknots from other coronoviruses.
TextSentencer_T172 27090-27322 Sentence denotes An answer to whether loop IIs in other coronoviruses need proper folding and to what extent they can accommodate sequence changes without loss of frameshifting requires more extensive studies of pseudoknots from other coronoviruses.
TextSentencer_T173 27323-27499 Sentence denotes However, it is clear that if such requirements exist they are highly diverse among coronaviruses as evident from the lack of conservation seen in comparative sequence analysis.
TextSentencer_T173 27323-27499 Sentence denotes However, it is clear that if such requirements exist they are highly diverse among coronaviruses as evident from the lack of conservation seen in comparative sequence analysis.
TextSentencer_T174 27500-27600 Sentence denotes Another feature found in this study is the importance of an unpaired GU tandem at the stem junction.
TextSentencer_T174 27500-27600 Sentence denotes Another feature found in this study is the importance of an unpaired GU tandem at the stem junction.
TextSentencer_T175 27601-27849 Sentence denotes Nucleotides at the equivalent position in IBV are base paired and disrupting the two base pairs at the top of stem I significantly reduced frameshifting efficiency, although the identity of the base pair was less important (Brierley et al., 1991) .
TextSentencer_T175 27601-27849 Sentence denotes Nucleotides at the equivalent position in IBV are base paired and disrupting the two base pairs at the top of stem I significantly reduced frameshifting efficiency, although the identity of the base pair was less important (Brierley et al., 1991) .
TextSentencer_T176 27850-28136 Sentence denotes Alterations in the SARS-CoV loop I which created base pairing potential with the unpaired GU or changing the unpaired G:U to A:C significantly impaired frameshifting levels, illustrating the importance of these two unpaired nucleotides for the frameshift stimulation by this pseudoknot.
TextSentencer_T176 27850-28136 Sentence denotes Alterations in the SARS-CoV loop I which created base pairing potential with the unpaired GU or changing the unpaired G:U to A:C significantly impaired frameshifting levels, illustrating the importance of these two unpaired nucleotides for the frameshift stimulation by this pseudoknot.
TextSentencer_T177 28137-28230 Sentence denotes These unpaired GU tandem nucleotides exist in about half of the pseudoknots shown in Fig. 1 .
TextSentencer_T177 28137-28230 Sentence denotes These unpaired GU tandem nucleotides exist in about half of the pseudoknots shown in Fig. 1 .
TextSentencer_T178 28231-28375 Sentence denotes Additional structural studies will be required to determine the role these two nucleotides play in obtaining the active pseudoknot conformation.
TextSentencer_T178 28231-28375 Sentence denotes Additional structural studies will be required to determine the role these two nucleotides play in obtaining the active pseudoknot conformation.
TextSentencer_T179 28376-28526 Sentence denotes Despite the sequence similarity of the SARS-CoV and IBV frameshift stimulatory elements, clearly they have a number of unique distinguishing features.
TextSentencer_T179 28376-28526 Sentence denotes Despite the sequence similarity of the SARS-CoV and IBV frameshift stimulatory elements, clearly they have a number of unique distinguishing features.
TextSentencer_T180 28527-28655 Sentence denotes In addition, we have shown that, unlike IBV, RNA in loop II of the SARS-CoV pseudoknot plays an important role in frameshifting.
TextSentencer_T180 28527-28655 Sentence denotes In addition, we have shown that, unlike IBV, RNA in loop II of the SARS-CoV pseudoknot plays an important role in frameshifting.
TextSentencer_T181 28656-28809 Sentence denotes In the context of a large loop II, this region needs to form a secondary structure with unpaired As in order to maintain frameshift stimulating activity.
TextSentencer_T181 28656-28809 Sentence denotes In the context of a large loop II, this region needs to form a secondary structure with unpaired As in order to maintain frameshift stimulating activity.
TextSentencer_T182 28810-28969 Sentence denotes In SARS-CoV, the stem loop structure, and bulged As likely play a role in the proper orientation of loop II relative to other components of the RNA pseudoknot.
TextSentencer_T182 28810-28969 Sentence denotes In SARS-CoV, the stem loop structure, and bulged As likely play a role in the proper orientation of loop II relative to other components of the RNA pseudoknot.
TextSentencer_T183 28970-29033 Sentence denotes Sequences of viral genomes were extracted from Refseq database.
TextSentencer_T183 28970-29033 Sentence denotes Sequences of viral genomes were extracted from Refseq database.
TextSentencer_T184 29034-29236 Sentence denotes Accession numbers are: SARS -NC _ 004718, IBV -NC _ 001451, MHV -NC _ 001846, HCV229E -NC _ 002 645, TGV -NC _ 002306, Bovine -NC _ 003045, PEDV -NC _ 003436, HCVOC43 -NC _ 005147, HCVNL63 -NC _ 005831.
TextSentencer_T184 29034-29236 Sentence denotes Accession numbers are: SARS -NC _ 004718, IBV -NC _ 001451, MHV -NC _ 001846, HCV229E -NC _ 002 645, TGV -NC _ 002306, Bovine -NC _ 003045, PEDV -NC _ 003436, HCVOC43 -NC _ 005147, HCVNL63 -NC _ 005831.
TextSentencer_T185 29237-29282 Sentence denotes For abbreviations used see legend to Fig. 1 .
TextSentencer_T185 29237-29282 Sentence denotes For abbreviations used see legend to Fig. 1 .
TextSentencer_T186 29283-29397 Sentence denotes Multiple alignments of viral sequences were generated using ClustalW (Thompson et al., 1994) and refined manually.
TextSentencer_T186 29283-29397 Sentence denotes Multiple alignments of viral sequences were generated using ClustalW (Thompson et al., 1994) and refined manually.
TextSentencer_T187 29398-29736 Sentence denotes Complementary oligonucleotides, to make constructs containing the sequences listed below, were synthesized at the University of Utah DNA/Peptide Core Facility so that when annealed they would have appropriate ends to ligate into either the SalI/BamHI or the SalI/SacI sites of the dual luciferase vector, p2luc (Grentzmann et al., 1998) .
TextSentencer_T187 29398-29736 Sentence denotes Complementary oligonucleotides, to make constructs containing the sequences listed below, were synthesized at the University of Utah DNA/Peptide Core Facility so that when annealed they would have appropriate ends to ligate into either the SalI/BamHI or the SalI/SacI sites of the dual luciferase vector, p2luc (Grentzmann et al., 1998) .
TextSentencer_T188 29737-29782 Sentence denotes All dual luciferase constructs were verified.
TextSentencer_T188 29737-29782 Sentence denotes All dual luciferase constructs were verified.
TextSentencer_T189 29783-29789 Sentence denotes SARSWT
TextSentencer_T189 29783-29789 Sentence denotes SARSWT
TextSentencer_T190 29790-29951 Sentence denotes The human embryonic kidney cell line, HEK 293, was obtained from ATCC and maintained as previously described (Howard et al., 2000) in the absence of antibiotics.
TextSentencer_T190 29790-29951 Sentence denotes The human embryonic kidney cell line, HEK 293, was obtained from ATCC and maintained as previously described (Howard et al., 2000) in the absence of antibiotics.
TextSentencer_T191 29952-30050 Sentence denotes Cells used in these studies were subcultured at 70% confluence and used between passages 7 and 15.
TextSentencer_T191 29952-30050 Sentence denotes Cells used in these studies were subcultured at 70% confluence and used between passages 7 and 15.
TextSentencer_T192 30051-30232 Sentence denotes Cells were transfected using Lipofectamine 2000 reagent (Invitrogen), using the One-day protocol in which suspension cells are added directly to the DNA complexes in 96-well plates.
TextSentencer_T192 30051-30232 Sentence denotes Cells were transfected using Lipofectamine 2000 reagent (Invitrogen), using the One-day protocol in which suspension cells are added directly to the DNA complexes in 96-well plates.
TextSentencer_T193 30233-30374 Sentence denotes 25 ng DNA and 0.2 Al Lipofectamine 2000/well in 25 Al Opti-Mem (Gibco) were incubated and plated in opaque 96-well half-area plates (Costar).
TextSentencer_T193 30233-30374 Sentence denotes 25 ng DNA and 0.2 Al Lipofectamine 2000/well in 25 Al Opti-Mem (Gibco) were incubated and plated in opaque 96-well half-area plates (Costar).
TextSentencer_T194 30375-30482 Sentence denotes Cells were trypsinized, washed and added at a concentration of 4 Â 10 4 cells/ well in 50 Al DMEM, 10% FBS.
TextSentencer_T194 30375-30482 Sentence denotes Cells were trypsinized, washed and added at a concentration of 4 Â 10 4 cells/ well in 50 Al DMEM, 10% FBS.
TextSentencer_T195 30483-30645 Sentence denotes Transfected cells were incubated overnight at 378 in 5% CO 2 , then 75 Al DMEM, 10% FBS were added to each well, and the plates were incubated an additional 48 h.
TextSentencer_T195 30483-30645 Sentence denotes Transfected cells were incubated overnight at 378 in 5% CO 2 , then 75 Al DMEM, 10% FBS were added to each well, and the plates were incubated an additional 48 h.
TextSentencer_T196 30646-30742 Sentence denotes Luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega).
TextSentencer_T196 30646-30742 Sentence denotes Luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega).
TextSentencer_T197 30743-30819 Sentence denotes Relative light units were measured on an MLX microplate luminometer (Dynex).
TextSentencer_T197 30743-30819 Sentence denotes Relative light units were measured on an MLX microplate luminometer (Dynex).
TextSentencer_T198 30820-30958 Sentence denotes Transfected cells were lysed in 12.5 Al lysis buffer and light emission was measured following injection of 50 Al of luminescence reagent.
TextSentencer_T198 30820-30958 Sentence denotes Transfected cells were lysed in 12.5 Al lysis buffer and light emission was measured following injection of 50 Al of luminescence reagent.
TextSentencer_T199 30959-31206 Sentence denotes Frameshifting was calculated by comparing firefly/renilla luciferase ratios of experimental constructs with those of control constructs: (firefly experimental RLUs / renilla experimental RLUs) / (firefly control RLUs / renilla control RLUs) Â 100.
TextSentencer_T199 30959-31206 Sentence denotes Frameshifting was calculated by comparing firefly/renilla luciferase ratios of experimental constructs with those of control constructs: (firefly experimental RLUs / renilla experimental RLUs) / (firefly control RLUs / renilla control RLUs) Â 100.
TextSentencer_T200 31207-31374 Sentence denotes The total number of independent experiments for each construct varies between 3 and 10, in each experiment, 3 independent data points were obtained for each construct.
TextSentencer_T200 31207-31374 Sentence denotes The total number of independent experiments for each construct varies between 3 and 10, in each experiment, 3 independent data points were obtained for each construct.
TextSentencer_T201 31375-31446 Sentence denotes In total between 9 and 30 data points were obtained for each construct.
TextSentencer_T201 31375-31446 Sentence denotes In total between 9 and 30 data points were obtained for each construct.
TextSentencer_T202 31447-31751 Sentence denotes Frameshift values were then standardized to the wildtype frameshift levels in that experiment, and for each construct they are as a percentage of the wildtype ((firefly experimental RLUs / renilla experimental RLUs) / (firefly control RLUs / renilla control RLUs) Â 100) / percent wildtype frameshifting.
TextSentencer_T202 31447-31751 Sentence denotes Frameshift values were then standardized to the wildtype frameshift levels in that experiment, and for each construct they are as a percentage of the wildtype ((firefly experimental RLUs / renilla experimental RLUs) / (firefly control RLUs / renilla control RLUs) Â 100) / percent wildtype frameshifting.
TextSentencer_T203 31752-31844 Sentence denotes For each construct all data points were then averaged and the standard deviation calculated.
TextSentencer_T203 31752-31844 Sentence denotes For each construct all data points were then averaged and the standard deviation calculated.
TextSentencer_T204 31845-31944 Sentence denotes Data points which fell greater than 2 standard deviations from the mean were discarded as outliers.
TextSentencer_T204 31845-31944 Sentence denotes Data points which fell greater than 2 standard deviations from the mean were discarded as outliers.
TextSentencer_T205 31945-32238 Sentence denotes A mammalian expression vector producing a triple tagged (GST, Maltose Binding Protein (MBP), and 6 Histidine) tagged fusion protein was constructed as follows: pCMV Sport-Bgal (Invitrogen) was digested with Pst1 and HindIII and purified by agarose gel electrophoresis to remove the B-gal gene.
TextSentencer_T205 31945-32238 Sentence denotes A mammalian expression vector producing a triple tagged (GST, Maltose Binding Protein (MBP), and 6 Histidine) tagged fusion protein was constructed as follows: pCMV Sport-Bgal (Invitrogen) was digested with Pst1 and HindIII and purified by agarose gel electrophoresis to remove the B-gal gene.
TextSentencer_T206 32239-32560 Sentence denotes A DNA fragment containing Pst1 and HindIII restriction sites, GST, MBP and 6 Histidine tags was produced by PCR amplification from plasmid GST-MBP-6xHis described in (Herr et al., 2001) using the primers; GAAGGCCTGCAGGTCAC-CATGTCGTTTTCCCCTATACTAGGTTATTGG, and G C G A C G G C A A G C T T TAT TA AT G AT G AT G AT-GATGGTG.
TextSentencer_T206 32239-32560 Sentence denotes A DNA fragment containing Pst1 and HindIII restriction sites, GST, MBP and 6 Histidine tags was produced by PCR amplification from plasmid GST-MBP-6xHis described in (Herr et al., 2001) using the primers; GAAGGCCTGCAGGTCAC-CATGTCGTTTTCCCCTATACTAGGTTATTGG, and G C G A C G G C A A G C T T TAT TA AT G AT G AT G AT-GATGGTG.
TextSentencer_T207 32561-32660 Sentence denotes This fragment was cloned into the PstI and HindIII sites of pCMV Sport Bgal to produce, pSport GMH.
TextSentencer_T207 32561-32660 Sentence denotes This fragment was cloned into the PstI and HindIII sites of pCMV Sport Bgal to produce, pSport GMH.
TextSentencer_T208 32661-33005 Sentence denotes The SARS frameshift cassette was inserted into the BamHI and EcoRI sites located between the GST and MBP genes by annealing synthetic oligonucleotides such that the sequence located between the restriction sites is derived entirely from SARS-CoV; CGTTTTTAAACGGGTTT-GCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGG-CACAGGCACTAGTACTGATGTCGTCTACAGGG-CTTTTGATAT.
TextSentencer_T208 32661-33005 Sentence denotes The SARS frameshift cassette was inserted into the BamHI and EcoRI sites located between the GST and MBP genes by annealing synthetic oligonucleotides such that the sequence located between the restriction sites is derived entirely from SARS-CoV; CGTTTTTAAACGGGTTT-GCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGG-CACAGGCACTAGTACTGATGTCGTCTACAGGG-CTTTTGATAT.
TextSentencer_T209 33006-33110 Sentence denotes The resulting plasmid pSport GMH SARS1 was utilized in the Freestyle 293 expression system (Invitrogen).
TextSentencer_T209 33006-33110 Sentence denotes The resulting plasmid pSport GMH SARS1 was utilized in the Freestyle 293 expression system (Invitrogen).
TextSentencer_T210 33111-33247 Sentence denotes 90 ml of HEK293 F suspension cells were transfected with pSport GMH SARS1 using 293fectin (Invitrogen) as described by the manufacturer.
TextSentencer_T210 33111-33247 Sentence denotes 90 ml of HEK293 F suspension cells were transfected with pSport GMH SARS1 using 293fectin (Invitrogen) as described by the manufacturer.
TextSentencer_T211 33248-33373 Sentence denotes After 48 h of incubation, the cells were lysed in PBS + 0.5% trition X-100 + protease inhibitors by 3Â Dounce homogenization.
TextSentencer_T211 33248-33373 Sentence denotes After 48 h of incubation, the cells were lysed in PBS + 0.5% trition X-100 + protease inhibitors by 3Â Dounce homogenization.
TextSentencer_T212 33374-33516 Sentence denotes The GST-SARS-MBP-6XHis fusion protein was purified by sequential passage over Glutathione sepharose 4B (Amersham) and Ni-NTA Agarose (Qiagen).
TextSentencer_T212 33374-33516 Sentence denotes The GST-SARS-MBP-6XHis fusion protein was purified by sequential passage over Glutathione sepharose 4B (Amersham) and Ni-NTA Agarose (Qiagen).
TextSentencer_T213 33517-33647 Sentence denotes The final product was concentrated and washed with ultrapure water 3Â using a centrifugal filter device, Ultrafree-15 (Millipore).
TextSentencer_T213 33517-33647 Sentence denotes The final product was concentrated and washed with ultrapure water 3Â using a centrifugal filter device, Ultrafree-15 (Millipore).
TextSentencer_T214 33648-33733 Sentence denotes The final product was digested with PreScission protease (AP biotech) as recommended.
TextSentencer_T214 33648-33733 Sentence denotes The final product was digested with PreScission protease (AP biotech) as recommended.
TextSentencer_T215 33734-33821 Sentence denotes Final desalting for mass spectrometry was performed using a C18 P10 Ziptip (Millipore).
TextSentencer_T215 33734-33821 Sentence denotes Final desalting for mass spectrometry was performed using a C18 P10 Ziptip (Millipore).
TextSentencer_T216 33822-34015 Sentence denotes Prior to loading the protein sample onto the Ziptip, the Ziptip was washed with 50 Al of acetonitrile containing 1% formic acid, then equilibrated with 20 Al of 15% acetonitrile (and 0.5% TFA).
TextSentencer_T216 33822-34015 Sentence denotes Prior to loading the protein sample onto the Ziptip, the Ziptip was washed with 50 Al of acetonitrile containing 1% formic acid, then equilibrated with 20 Al of 15% acetonitrile (and 0.5% TFA).
TextSentencer_T217 34016-34120 Sentence denotes Methanol and TFA were added to the protein sample to a final concentration of 5% and 0.5%, respectively.
TextSentencer_T217 34016-34120 Sentence denotes Methanol and TFA were added to the protein sample to a final concentration of 5% and 0.5%, respectively.
TextSentencer_T218 34121-34211 Sentence denotes The protein sample was then loaded onto the Ziptip by pipetting up and down several times.
TextSentencer_T218 34121-34211 Sentence denotes The protein sample was then loaded onto the Ziptip by pipetting up and down several times.
TextSentencer_T219 34212-34345 Sentence denotes To desalt the protein, the Ziptip was washed with 40 Al of 15% acetonitrile (and 0.5% TFA) then washed with 40 Al of nano-pure water.
TextSentencer_T219 34212-34345 Sentence denotes To desalt the protein, the Ziptip was washed with 40 Al of 15% acetonitrile (and 0.5% TFA) then washed with 40 Al of nano-pure water.
TextSentencer_T220 34346-34522 Sentence denotes The protein was eluted from the Ziptip with 2 aliquots of 1 Al of 55% acetonitrile (and 1% formic acid) followed by 2 aliquots of 1 Al of 80% acetonitrile (and 1% formic acid).
TextSentencer_T220 34346-34522 Sentence denotes The protein was eluted from the Ziptip with 2 aliquots of 1 Al of 55% acetonitrile (and 1% formic acid) followed by 2 aliquots of 1 Al of 80% acetonitrile (and 1% formic acid).
TextSentencer_T221 34523-34581 Sentence denotes The aliquots were combined for mass spectrometry analysis.
TextSentencer_T221 34523-34581 Sentence denotes The aliquots were combined for mass spectrometry analysis.
TextSentencer_T222 34582-34660 Sentence denotes The protein was introduced into the mass spectrometer by infusion at 3 Al/min.
TextSentencer_T222 34582-34660 Sentence denotes The protein was introduced into the mass spectrometer by infusion at 3 Al/min.
TextSentencer_T223 34661-34791 Sentence denotes Mass measurements were performed with a Quattro II mass spectrometer (Micromass, Inc.) using positive-ion electrospray ionization.
TextSentencer_T223 34661-34791 Sentence denotes Mass measurements were performed with a Quattro II mass spectrometer (Micromass, Inc.) using positive-ion electrospray ionization.
TextSentencer_T224 34792-34955 Sentence denotes The instrument was scanned from 800 to 1400 kDa in 4 s at unit resolution with 3 kV spray voltage and 55 eV cone voltage and scans were accumulated for 1 to 2 min.
TextSentencer_T224 34792-34955 Sentence denotes The instrument was scanned from 800 to 1400 kDa in 4 s at unit resolution with 3 kV spray voltage and 55 eV cone voltage and scans were accumulated for 1 to 2 min.
TextSentencer_T225 34956-35133 Sentence denotes All spectra were acquired using Masslynx software (Micromass) and multiply-charged ion series were processed into molecular-mass spectra using MaxEnt software (Micromass, Inc.).
TextSentencer_T225 34956-35133 Sentence denotes All spectra were acquired using Masslynx software (Micromass) and multiply-charged ion series were processed into molecular-mass spectra using MaxEnt software (Micromass, Inc.).