Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nayak, A. Articles by Belsham, G. J. Search for Related Content PubMed PubMed Citation Articles by Nayak, A. Articles by Belsham, G. J.

 Previous Article  |  Next Article 

Journal of Virology, October 2006, p. 9865-9875, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00561-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of RNA Structure and RNA Binding Activity of Foot-and-Mouth Disease Virus 3C Protein in VPg Uridylylation and Virus Replication

Arabinda Nayak,^1, Ian G. Goodfellow,² Kathryn E. Woolaway,^1, James Birtley,³ Stephen Curry,³ and Graham J. Belsham^1*

BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, United Kingdom,¹ Department of Virology, Faculty of Medicine, Imperial College, St Mary's Campus, London W2 1PG, United Kingdom,² Biophysics Section, Blackett Laboratory, Imperial College, South Kensington Campus, London SW7 2AZ, United Kingdom³

Received 17 March 2006/ Accepted 7 July 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3D^pol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B₃3C and 3B₁₂₃3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The family Picornaviridae includes important pathogens of humans and other mammals, e.g., poliovirus (PV), human rhinovirus (HRV), and foot-and-mouth disease virus (FMDV). Picornavirus genomes are positive-sense RNA molecules of about 8 kb that function both as mRNAs (to produce the virus-encoded proteins) and as templates for the production of new RNA transcripts (4, 32). The genomic RNA is uncapped (cf. eukaryotic cellular mRNAs) but is linked at its 5' terminus to the virus-encoded peptide VPg (or 3B). All picornavirus genomes have a 5'-terminal sequence of VPgpUpU... , and this is derived from the use of VPg as a peptide primer for the synthesis of the RNA. Attachment of this peptide to the RNA occurs via a Tyr (Y) residue and is performed by the viral RNA polymerase (3D^pol). For PV, HRV, and FMDV, it has been shown that the uridylylation of VPg can be achieved in vitro using purified VPg (as a synthetic peptide) with 3D^pol, the precursor 3CD, UTP, and an RNA template that includes a stem-loop structure frequently termed a cis-acting replication element (cre) (15, 29, 33). This reaction produces VPgpU and VPgpUpU [collectively termed VPgpU(pU)]. It had been anticipated that such primers would function for the formation of both positive- and negative-sense RNAs (32). Unexpectedly, within an in vitro replication system, experiments show that the PV cre is not required for negative-sense RNA synthesis (19, 26, 28). However, recently it has been reported that certain mutations in the coxsackievirus B3 cre have affected both positive- and negative-strand RNA synthesis (43). Thus, the mechanism involved in the initiation of negative-sense RNA synthesis is still not resolved.

Uniquely, FMDV encodes and uses three distinct VPg peptides, which are 23 or 24 amino acids in length (21). Each of the FMDV VPgs can be uridylylated in vitro, although VPg3 (3B3) seems to be the most efficient substrate (29). It is interesting that modification of just the VPg3 coding sequence within the context of the full-length (FL) infectious cDNA resulted in the production of a noninfectious RNA transcript (13).

The first cre identified within picornavirus RNA was within the sequence encoding the 1D (VP1) protein of HRV type 14 (HRV-14). This element has been shown to be required in the form of RNA for replication of the viral RNA (24, 25). Subsequently, similar elements have been identified in other picornavirus genomes (15, 17, 22). Each of these cre structures (about 50 to 60 nucleotides [nt] in length) includes a conserved sequence motif, AAACA, located within a loop at the end of a stable stem. These elements occur in different locations within various picornavirus genomes; the cre structures from HRV-14, HRV-2, cardioviruses, and PV are within the coding regions for 1D, 2A, 1B, and 2C, respectively (15, 22, 25, 33). Uniquely, the FMDV cre is located within the 5' untranslated region (5' UTR) of the RNA (23), just upstream of the internal ribosome entry site (IRES). The cre structures can be moved without blocking function (18, 23).

The A¹A²A³CA motif within the cre acts as the template for the uridylylation of VPg, and within this motif, the A¹ nucleotide is absolutely essential for the reaction, while modification of either A² or A³ severely inhibits it (35). It has been shown that a "slideback" mechanism is involved in the uridylylation of the PV VPg and that the A¹ nucleotide acts as the template for the addition of both uridyl groups to the peptide. Data on the requirements for this motif within the FMDV element are consistent with the same mechanism (29). Recently, the structure of the cre from HRV has been determined using nuclear magnetic resonance (40), but surprisingly, the AAACA motif was not very exposed. Genetic studies have indicated that the FMDV cre can function in trans (41), and hence, it has been suggested that this element should be termed a 3B-uridylylation site (bus). The PV cre can also function in trans within in vitro assays (19), and recent data from Crowder and Kirkegaard (11) showed that mutations within the PV cre can inhibit PV replication in a trans-dominant manner within cells.

The nature of the essential role of 3CD in the uridylylation reaction is not entirely clear. It is well known that PV 3CD can bind to RNA and is able to interact with the 5'-terminal cloverleaf structure on PV RNA (1) and also specifically with the PV cre (48). Specific residues, within a conserved KFRDI (Lys-Phe-Arg-Asp-Ile) sequence that are required for interaction of the picornavirus 3C proteins with RNA have been identified (1, 5, 27, 44). It has also been shown that the role of PV 3CD in the uridylylation reaction can be fulfilled by 3C alone, although the reaction is less efficient (31). The structures of several picornavirus 3C proteins have been determined by X-ray crystallography, including that encoded by FMDV (5, 7, 27), but currently, no structure for a 3CD protein has been reported. The RNA binding regions of the PV and hepatitis A virus (HAV) 3C proteases have been mapped to sequences on the face of the molecule opposite from the catalytic active site (5, 27, 33, 47). Modification of both R84 and I86 within the K⁸²FRDI⁸⁶ motif of the PV 3C protease blocked the ability of the protein to bind the "cloverleaf" RNA and to support PV VPg uridylylation (1, 33). We have now explored the roles of sequences within the FMDV 3C protein that are required for the uridylylation of FMDV VPg in vitro. We have identified individual amino acids on the surface of the FMDV 3C protein that are critical for uridylylation and that also modify the protein's interaction with the cre/bus. In addition, we have investigated the nature of the RNA template involved in the uridylylation reaction and have shown that the presence of the FMDV IRES within the RNA transcript enhances the template activity of the adjacent cre/bus structure.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production and modification of plasmids. The plasmids containing FMDV O1Kaufbeuren (O1K) cDNA sequences, namely, pUb-3D^pol, pQE30/3CD^pro(C163G), pGC-cre, and pT7S3, have been described previously (12, 29). Similarly, the plasmid pETm11-3C, which encodes the 3C protease from FMDV A10₆₁, has also been described previously (6, 7). Mutations producing single-amino-acid substitutions in this 3C protease were introduced by QuikChange mutagenesis (Strategene) using the primers listed in Table 1. A region (nt 403 to 1120) of the FMDV 5' UTR (O1Kaufbeuren) was amplified using primers poly(C)-down-For and IRES/REV and cloned into pT7blue (Novagen) to create pT7(PK+cre+IRES). The manipulation, analysis, and growth of plasmids were achieved using standard techniques (37).

View this table: [in this window] [in a new window]

TABLE 1. Oligonucleotide primers used in this study

Expression and purification of FMDV 3D^pol. Expression and purification of the untagged FMDV 3D^pol was achieved by production and cleavage of ubiquitin-tagged FMDV 3D^pol by coexpression with the Ubp2 protease as described previously (16, 29).

Production and purification of FMDV 3C^pro(C163G). The coding sequence of O1K 3C^pro was amplified by PCR using oligonucleotides O1K/3CFOR and O1K/3CREV, with BamHI and HindIII restriction sites, respectively (Table 1), using pQE30/3CD^pro(C163G) as a template (29). The fragment was inserted into the pT7-blue vector (Novagen), and the resulting plasmid was digested with BamHI and HindIII; the released insert was ligated between the same sites of the pQE30 vector. The expression of this protein and the protease-inactive forms of His-tagged 3CD(C163G) and the 3C(A10₆₁) proteins plus their derivatives were achieved and purified as described previously (6, 7, 29). The 3C from the A10₆₁ strain of FMDV has amino acid substitutions C95K, C142S, and C163A to inactivate the proteolytic activity and enhance its solubility (6, 7).

Production of FMDV 3B₃3C and 3B₁₂₃3C. For expression of the FMDV nonstructural precursor proteins 3B₃3C and 3B₁₂₃3C in the ubiquitin system (16), the cDNA fragment was amplified by PCR using the appropriate primers (Table 1) and cloned into the pT7-blue vector. Modification within the 3C protease sequence to change the codon for residue C163 to Gly in the pT7-3B₃3C and pT7-3B₁₂₃3C constructs was carried out using the QuikChange site-directed mutagenesis protocol as described previously (29), using appropriate primers (listed in Table 1). The modified fragments were excised from the pT7-blue clones using SacII and BamHI and cloned into a similarly digested pET26b-Ub vector. The presence of the desired mutation was confirmed by sequencing. Coexpression with the ubiquitin protease and purification of the native 3B₃3C and 3B₁₂₃3C proteins was performed essentially as described for the native 3D^pol (16, 29). The phosphocellulose column-purified fractions were directly loaded onto a 1-ml Hi-trap SP Sepharose column (Amersham Bioscience), eluted by gradient elution, and dialyzed using buffer A (50 mM Tris, pH 8.0, 20% glycerol, 10 mM ß-mercaptoethanol, and 0.1% NP-40) with 750 or 500 mM NaCl, respectively.

Construction of infectious cDNAs for FMDV encoding single-amino-acid substitutions within the 3C protease. A PCR fragment (1,750 bp) encompassing the FMDV 3C coding sequence (O1K), along with the preexisting BclI and BlpI restriction sites, was amplified using primers O1KBclI and O1KBlpI (Table 1) and ligated into the pT7-blue vector. The resultant clone (pT7-BB) was modified by site-directed mutagenesis (QuikChange mutagenesis kit; Stratagene) to encode serines at the codons for R92, R95, and R97 using appropriate primers (Table 1). Positive clones were grown using a dam and dcm mutant Escherichia coli strain (ER2925 from New England Biotechnology). The purified DNA was digested with BclI and BlpI to release an insert of 1,729 bp and introduced into the similarly digested FMDV infectious cDNA clone pT7S3 (12). The presence of each mutation in the full-length cDNA was confirmed by sequencing.

Electroporation of FMDV RNA into BHK cells. Wild-type (WT) or mutant full-length FMDV RNA transcripts (ca. 4 µg) were prepared using a Megascript kit (Ambion) containing T7 RNA polymerase and were added to BHK 21 cells (2 x 10⁶) suspended in siPORT siRNA Electroporation Buffer (Ambion) (0.75 ml), which were shocked (1 kV; 0.6 ms; two pulses) using an Electro Square Porator T820 (BTX Electronics). Following incubation at room temperature for 10 min, the cells were transferred to a 25-cm² flask containing 5 ml of virus growth medium with 1% fetal calf serum and incubated for 8 h at 37°C. The recovered virus was released by freeze-thawing, and the yield was determined by plaque assay on BHK cells. The plaques were visualized after 40 to 48 h, using methylene blue with 4% formaldehyde in phosphate-buffered saline.

Uridylylation assays. The synthesis of VPgpU(pU) was measured essentially as described previously (18, 29, 33). Unless otherwise indicated, the reaction mixtures contained 50 mM HEPES (pH 7.5), glycerol (8%), MgCl₂ (2 mM), 3B3/VPg3 (12.5 µM), cre/bus RNA transcripts (1 µM), [-³²P]UTP (0.75 µCi; Amersham Bioscience), UTP (10 µM), His-3CD(C163G) (1 µM), and 3D^pol (1 µM). The final NaCl concentration in the reaction mixture (from the protein solutions) was kept at 6.25 mM. Reaction mixtures were incubated for 30 min at 30°C and analyzed by Tris-tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 14% polyacrylamide. The gels were dried and autoradiographed. The products were quantitated with a PhosphorImager (Molecular Imager FX; Bio-Rad) to determine the level of [³²P]UMP incorporation. Other RNA transcripts were produced when required using T7 RNA polymerase on appropriately linearized plasmids.

Preparation of BHK S10 extracts. Monolayers of BHK cells (five 175-cm² flasks) were harvested using a cell scraper, washed in phosphate-buffered saline, resuspended in cold hypotonic buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM magnesium acetate, 1 mM dithiothreitol [DTT]) (5 ml), and allowed to swell on ice for 15 min. The cells were lysed with a Dounce homogenizer (40 strokes) and centrifuged (5,000 x g for 5 min) to remove the cellular debris and nuclei. Cytoplasmic extract (S10) was prepared by centrifuging the postnuclear fraction at 10,000 x g for 15 min at 4°C. The supernatant was dialyzed for 3 h against 40 mM HEPES, pH 8.0, 120 mM potassium acetate, 5.5 mM magnesium acetate, 10 mM KCl, and 6 mM DTT; aliquoted; and stored at –70°C.

Cell-free translation of FMDV RNA in BHK S10 extracts. In vitro translation reactions were performed using mixtures (25 µl) containing 70% BHK S10, 1 µg of FMDV RNA transcripts (wild type or mutant), 50 mM HEPES (pH 8.0), 50 mM potassium acetate, 1.0 mM ATP, 0.25 mM GTP, 0.25 mM CTP, 0.25 mM UTP, 5 mM creatine phosphate, 50 µg/ml creatine phosphokinase (Sigma), 800 U of RNasin/ml in the presence of [³⁵S]methionine. The reaction mixtures were incubated for 90 min at 30°C and analyzed by SDS-PAGE and autoradiography.

RNA isolation, reverse transcription-PCR amplification, and sequencing. Total RNA from virus-infected BHK cells was extracted with Trizol (Invitrogen, Life Technologies). Viral cDNA was made using SuperScript III Reverse Transcriptase (Invitrogen) with selective viral primers and amplified using PCR. The products were purified from agarose gels and sequenced using the CEQ 8000 Genetic Analysis System (Beckman Coulter).

RNA-protein interaction assays. The binding of WT and mutant 3C proteins to ³²P-labeled FMDV cre/bus RNA transcripts was analyzed using a filter binding assay (10, 49). ³²P-labeled RNA transcripts corresponding to the FMDV cre/bus were produced using a Maxiscript kit (Ambion) with pGC-cre (29) as a template with [-³²P]UTP. The reaction was performed using buffer conditions similar to those of the in vitro uridylylation assay (50 mM HEPES, pH 7.5, 8% glycerol, 2 mM magnesium chloride); total volume, 20 µl). The RNA (2.5 x 10⁴ cpm) was incubated with different concentrations of 3C protein (0.5 to 10 µM). Assays were performed for 20 min at 30°C and spotted onto Protran BA-85 nitrocellulose filters (Schleicher & Schuell). The filters were extensively washed with 50 mM HEPES, pH 7.5, 8% glycerol, 2 mM magnesium chloride and dried, and the bound RNA was measured using liquid scintillation. The percentage of RNA bound was calculated. UV cross-linking assays were performed using 0.4 µg of purified recombinant proteins [3C(wt), 3C(R92S), 3C(R95S), and 3C(R97S)] or bovine serum albumin with uniformly radiolabeled FMDV S-fragment RNA transcripts (2 x 10⁵ cpm) in 20 µl of 10 mM HEPES-KOH (pH 7.9), 25 mM KCl, 2 mM MgCl₂, 10% glycerol, 0.05% NP-40, 0.5 mM DTT, 10 µg tRNA. After a 30-min incubation on ice, the reaction mixtures were exposed to UV light for 30 min on ice. The samples were treated with 2 µl RNase cocktail (Ambion) for 30 min at 37°C, and the proteins were analyzed using 10% SDS-PAGE and autoradiography.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of the optimal RNA sequences required as the template for FMDV VPg uridylylation. In our previous studies (29), it was shown that the FMDV cre/bus transcript (ca. 50 nt), at relatively high concentration (1 µM), acts as an efficient template for VPg uridylylation; however, larger RNA transcripts (e.g., the FL RNA) were significantly more efficient. To define the optimal RNA sequences required for this reaction, a series of RNA transcripts were prepared, including different regions of the FMDV 5' UTR; they are indicated in Fig. 1A. Each transcript was used at 35 nM within a standard uridylylation assay using VPg3 as a substrate (Fig. 1B). As shown previously (29), the FL-RNA and the Wt-XbaI transcript were significantly more active than the short cre/bus transcript itself. Furthermore, the entire 5' UTR and the PK+cre+IRES transcript were even more efficient. However, the PK+cre transcript was not much better than the cre/bus transcript alone. These results suggested that the presence of the IRES sequences within the transcript significantly enhanced the template activity in this reaction. This probably reflects some modification of the structure or availability of the cre/bus in the presence of these additional sequences.

View larger version (21K): [in this window] [in a new window]

FIG. 1. The activity of the FMDV cre/bus as a template for VPg uridylylation is enhanced by the presence of IRES sequences. (A) Genome organization of FMDV and locations of RNA transcripts. Specific features within the 5' UTR, including the poly(C) tract, pseudoknots (PK), the cre/bus, and the IRES, are shown. The RNA transcripts each contained the cre/bus, as indicated by the thicker lines. (B) In vitro uridylylation assays were performed using VPg3 as a substrate in the presence of 3Dpol, 3CD, Mg2+, [-32P]UTP, and the indicated RNA transcripts (35 nM in each case). Reaction mixtures were incubated at 30°C for 30 min. The products were analyzed using Tris-tricine gels and autoradiography, as described previously (29). Quantitation of 32P-labeled VPgpU(pU) was achieved using a phosphorimager.

FMDV 3C can substitute for FMDV 3CD for the uridylylation of FMDV VPg. In our previous studies, it was shown that the uridylylation of FMDV VPgs required UTP plus Mg²⁺ (or Mn²⁺) and was also dependent on the presence of each of the following FMDV components: 3D^pol with 3CD plus the RNA template incorporating the cre/bus (29). This closely mirrors the requirements for uridylylation of the PV or HRV-2 VPg (15, 33, 34). However, it should be noted that, in contrast to the PV system, the FMDV components do not significantly utilize poly(A) as a template even in the presence of Mn²⁺ (29). Since Pathak et al. (31) have demonstrated that the role of PV 3CD in this reaction can be fulfilled by 3C alone, we investigated whether the FMDV 3C protein could replace 3CD in the FMDV VPg uridylylation assay. The results obtained using the cre/bus transcript (ca. 50 nt) as a template showed that the 3C protein from either the type O or type A serotype of FMDV can substitute for 3CD (from serotype O) in this assay (Fig. 2A). To determine the relative efficiency of the reaction in the presence of 3C or 3CD, a series of reactions was performed using a range of 3C concentrations in the presence of VPg3 with the cre/bus transcript as a template (Fig. 2B). Under these conditions, it was found that even at 10 µM 3C, the efficiency of VPg uridylylation was only about 25% of that achieved with 1 µM 3CD (Fig. 2B). Furthermore, using the complete FMDV 5' UTR as the RNA template, which is much more efficient than using the cre/bus transcript (Fig. 1B), 3C was again less efficient than 3CD in supporting the reaction, but under these conditions, higher concentrations of the 3C protein inhibited the reaction (Fig. 2C).

View larger version (21K): [in this window] [in a new window]

FIG. 2. FMDV 3C protein can play the role of FMDV 3CD in VPg uridylylation. (A) In vitro uridylylation assays using the FMDV 3B3 (VPg3) peptide were performed using the WT FMDV cre/bus RNA transcript (1 µM) as a template in the presence of [-32P]UTP, Mg2+, FMDV 3Dpol alone or in the presence of His-tagged 3C (1 µM or 2.5 µM, as indicated) derived from FMDV strain O1K (solid bars), or A1061 (hatched bars), as indicated, and analyzed using Tris-tricine gels and autoradiography as described in the legend to Fig. 1. (B) In vitro uridylylation assays were performed as for panel A in the presence of either His-tagged 3CD (1 µM; strain O1K) or a range of concentrations of His-tagged 3C (O1K), as indicated, using the cre/bus transcript (1 µM) as a template. (C) In vitro uridylylation assays were performed as for panel B, except that the RNA template (35 nM) used was the complete FMDV 5' UTR (O1K).

Precursors of FMDV VPg, termed 3B₃3C and 3B₁₂₃3C, can be uridylylated in vitro. The VPg peptide is derived from the P3 precursor and can potentially be liberated from various different precursors, e.g., 3AB or 3BC. Since 3C can support the uridylylation of VPg, it seemed possible that 3BC would be a good substrate for this reaction even in the absence of added 3C or 3CD. To test this idea, two different precursors of the FMDV VPg were expressed in E. coli and purified to near homogeneity (Fig. 3A). The recombinant 3B₃3C and 3B₁₂₃3C precursor proteins (which lacked protease activity due to modification of a catalytic residue, C163G) were then assayed within in vitro uridylylation assays using the cre/bus transcript with 3D^pol alone or in the presence of added His-3CD. Both precursors were efficiently uridylylated, both in the presence and absence of 3CD (Fig. 3B and C). However, the addition of 3CD still stimulated the reactions (ca. three- to fivefold), consistent with the enhanced uridylylation of VPg in the presence of 3CD rather than just 3C (Fig. 2). The precursor 3B₃3C was a more efficient substrate than 3B₁₂₃3C, although neither was as good as the 3B₃ (VPg) peptide alone on an equimolar basis (Fig. 3D).

View larger version (47K): [in this window] [in a new window]

FIG. 3. Precursors of FMDV VPg, 3B33C and 3B1233C, can be efficiently uridylylated in vitro. (A) The precursor proteins 3B33C and 3B1233C were expressed in E. coli and purified to near homogeneity. Samples were analyzed by SDS-PAGE and stained with Coomassie blue; the stained gel is shown. (B and C) In vitro uridylylation reactions were performed as for Fig. 1 using the FMDV cre/bus (1 µM) as a template in the absence or presence of 3CD (as indicated) and using different concentrations of the precursor proteins 3B33C (B) or 3B1233C (C) as a substrate. The reactions were analyzed using Tris-tricine gels and autoradiography. (D) The 3B3 peptide and the precursor proteins were assayed in parallel within in vitro uridylylation assays, using equivalent concentrations of each substrate in the presence of the cre/bus template and 3CD. The autoradiograph is overexposed for uridylylation of the peptide substrate to permit the modification of the precursors to be observed.

Identification of a putative RNA binding site on the surface of the FMDV 3C protein. Individual residues within a conserved motif (KFR⁸⁴DI⁸⁶) of PV 3C that affect its binding to the cloverleaf at the 5' end of the PV genome have been identified (1). Residue R84 within this motif was found to be critical for RNA binding, and modification of R84 and I86 blocked the ability of PV 3CD to support VPg uridylylation (33). A comparison of the PV, HAV, HRV, and FMDV 3C sequences in this region is shown in Fig. 4. The KFRDI sequence is partially conserved as (R/K)VRDI⁹⁹ in FMDV; the PV residue R84 corresponds to R97 within the FMDV 3C sequence. Since the structure of the FMDV 3C protein has been determined recently (7), it is possible to locate this motif, and residue R97 in particular, on the surface of 3C. Consistent with observations of the PV and HAV 3C proteins (5, 27), this motif on the FMDV protein is located on the face of the protein opposite from the protease active site (Fig. 5). It is apparent that other positively charged residues are close to R97 on the surface of the FMDV 3C protein, including residues R92 and K/R95. Residues corresponding to R97 of FMDV are absolutely conserved in the 3C proteins of other FMDVs, PV, HRV, and HAV, while at the position of residue 95 in the FMDV 3C, there is either K or R (both positively charged) in each of these proteins. It is noteworthy that residue R92 in the FMDV 3C protein is also conserved in PV and HRV, but not in HAV. Note that in the original FMDV A10₆₁ incomplete cDNA, the 3C residue 95 was encoded as a Cys (C) but was mutated to Lys (K) (as found in other FMDV sequences) to increase the solubility of the protein (6, 7); it is not known whether the presence of a C residue at this position is compatible with virus viability.

View larger version (39K): [in this window] [in a new window]

FIG. 4. Alignment of picornavirus 3C protein sequences. Sections of the 3C amino acid sequences from multiple picornavirus genera were aligned using BIOEDIT. The conserved motif (K/R)(V/F)RDI implicated in RNA binding activity is boxed. Other highly conserved residues are also highlighted, in particular, FMDV R92 (arrow) and FMDV D84 (*), which is part of the catalytic triad.

View larger version (40K): [in this window] [in a new window]

FIG. 5. Identification of a potential RNA binding site on the surface of the FMDV 3C protein. (A) A surface representation of the structure of FMDV 3C protease (strain A1061) as determined by Birtley et al. (7), with the electrostatic potential of each residue shown. The positively charged residues are identified in blue, while the negatively charged residues are indicated in red. (B) A ribbon diagram showing the secondary-structure elements of the FMDV 3C protease. The view is the same as for panel A, and key residues (see the text) are marked by the presence of side chains. (C) Ribbon diagram of FMDV 3C showing the separation of catalytic and RNA binding residues of FMDV 3C. Note that this view is rotated through 90° compared to panel B.

Analysis of putative RNA binding residues in FMDV 3C. In order to test the roles of these basic residues for the uridylylation function, we generated three mutant 3C proteins in which residues R92, K95, and R97 were each replaced by an uncharged serine (S) residue. The WT and mutant His-tagged 3C proteins were each expressed in E. coli and purified to near homogeneity. Equal amounts of these proteins, as validated by Western blot analysis (Fig. 6A), were then tested in uridylylation assays in conjunction with 3D^pol and either the cre/bus transcript (Fig. 6B) or the entire FMDV 5' UTR (Fig. 6C) as the RNA template. In each case, the mutation had a very deleterious effect on uridylylation activity. The R97S mutant was almost completely inactive (<5% of the WT) in these assays, and the R92S mutant was also severely debilitated (ca. 6% of the WT). The K95S mutant was the most active mutant but still displayed only about 25% of WT activity.

View larger version (23K): [in this window] [in a new window]

FIG. 6. Identification of critical residues within FMDV 3C protein required for VPg uridylylation. (A) WT and mutant forms (R92S, K95S, and R97S) of the FMDV 3C protein (A1061) were expressed in E. coli, purified to homogeneity, and quantified. Equal amounts were analyzed by SDS-PAGE and immunoblotting using an anti-3C monoclonal antibody and detected using peroxidase-labeled anti-mouse immunoglobulin G. (B and C) The WT and mutant forms of FMDV 3C (strain A1061) were tested in uridylylation assays with FMDV 3Dpol, VPg3, Mg2+, and [-32P]UTP. The reaction mixtures contained either the cre/bus transcript (1 µM) (B) or the entire FMDV 5' UTR (35 nM) (C). In each case, the products were analyzed using Tris-tricine gels and autoradiography. Incorporation of UMP was quantified using a phosphorimager.

To confirm that these mutations in FMDV 3C resulted in a defect in RNA binding, the abilities of these proteins to bind ³²P-labeled cre/bus transcripts were determined using a filter binding assay (Fig. 7A). The R92S and R97S proteins were both significantly reduced in their abilities to bind this RNA transcript compared to the WT 3C protein. The K95S mutant was also deficient in cre/bus binding but was slightly more active than the other mutants. These results closely mirrored the abilities of these proteins to support the uridylylation of VPg (Fig. 6). In addition, the ability of the WT and mutant 3C proteins to bind a ³²P-labeled RNA transcript corresponding to the FMDV S fragment (Fig. 1) was analyzed using a UV cross-linking assay (Fig. 7B). The WT 3C protein was efficiently cross-linked to the RNA, but the mutant proteins interacted with the S fragment significantly less well; the results showed a pattern similar to that observed in the filter binding assay using the cre/bus transcript. The defect in uridylylation activity appears greater than the defect in RNA binding; this may reflect the nature of the assays or may suggest that these residues also have a specific role in VPg uridylyation.

View larger version (34K): [in this window] [in a new window]

FIG. 7. Analysis of residues within the FMDV 3C protein required for RNA-protein complex formation. (A) WT and mutant forms (R92S, K95S, and R97S) of FMDV 3C protein were used (as indicated) in filter binding assays using 32P-labeled cre/bus RNA transcripts as described in Materials and Methods. Bound RNA was quantified by scintillation counting. (B) 32P-labeled FMDV S-fragment transcripts were incubated with the indicated proteins and then exposed to UV light. Following RNase digestion, the samples were analyzed by SDS-PAGE and autoradiography.

Roles of RNA binding residues in FMDV 3C in virus replication. The codons for residues R92, R95, and R97 of the FMDV 3C were modified individually to encode serine residues within a full-length infectious cDNA of FMDV (O1K strain) termed pT7S3 (12). RNA transcripts were prepared and translated in an in vitro translation system using a BHK cell S10 lysate. The WT and mutant transcripts produced very similar patterns of polypeptides (Fig. 8A), indicating that normal polyprotein processing occurred; this showed that the proteolytic activity of the 3C protein was not greatly affected by any of these mutations. The full-length RNA transcripts were also introduced into BHK cells using electroporation. It was found that the R95S and R97S mutant transcripts were noninfectious; no plaques were observed from cell harvests prepared at 8 h postelectroporation. The R92S mutant transcripts did produce virus, but the yield of virus produced at 8 h postelectroporation was reduced about 10-fold compared to the WT transcript (Fig. 8B). Sequencing of reverse transcription-PCR products obtained from the rescued viruses indicated that they retained the R92S mutation (data not shown), but the presence of second-site mutations has not been examined. These results indicated the importance of each of these residues for efficient virus replication.

View larger version (91K): [in this window] [in a new window]

FIG. 8. RNA binding mutants within FMDV 3C are defective in virus replication. (A) Full-length RNA transcripts derived from the WT and mutant plasmids, as indicated, were translated in vitro using BHK S10 extract with [35S]Met. Samples were analyzed by SDS-PAGE and autoradiography. (B) RNA transcripts from the indicated plasmids were electroporated into BHK cells. After 8 h, the cells were harvested, and the virus produced was visualized using a plaque assay in BHK cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The uridylylation of VPg is the primary step in the process of picornavirus RNA replication. For optimal activity in vitro, the uridylylation of FMDV VPgs requires a combination of three different polypeptides (3D^pol, 3CD, and VPg), an RNA transcript including the cre/bus plus UTP and divalent metal ions (29). Similar results have been obtained using PV and HRV components (15, 33, 34). Intriguingly, the PV system is also able to produce VPgpU(pU) using a poly(A) template in the presence of Mn²⁺, and in recent studies, Arias et al. (3) have obtained a similar result for FMDV VPg. However under our conditions, which include a VPg concentration of 12.5 µM (over 10-fold lower than that used by Arias et al. [3]), we did not observe significant VPg uridylylation when using a poly(A) template (29). We have now shown that the 3D^pol from type C FMDV (kindly provided by A. Arias and E. Domingo) used for the structural studies (3, 14) exhibits characteristics very similar to those of the type O enzyme used in our functional studies, including a strong requirement for 3CD to uridylylate VPg when the cre/bus is used as a template in the presence of Mg²⁺ (data not shown).

The nature of the complexes formed between the different components of the uridylylation reaction is not yet established. However, for both the PV and FMDV proteins, it is now clear that the role of the 3CD protein (which lacks RNA polymerase activity) in this reaction can be functionally filled, albeit less efficiently, by the 3C protein alone (reference 31 and this work). Furthermore, the ability of the 3C protein to interact with RNA is essential for this role (reference 33 and this work). The complete complex, including 3C or 3CD, is required only when the cre/bus is used as the template, suggesting that 3C/3CD (or other precursor proteins) are involved in recognition of the RNA template. This is consistent with the demonstration that PV 3CD exhibits specific binding activity for the PV cre (48).

Structural studies of different picornavirus 3C proteins have shown that the binding site for RNA on 3C is on the face opposite from the residues that comprise the catalytic site of the protease. A conserved KFR⁸⁴DI⁸⁶ motif is important for the RNA binding activity of the PV and HAV 3C proteins; residues R84 and I86 within this motif have been shown to be critical (1, 33). The FMDV motif has a slightly different sequence: (R/K)VR⁹⁷DI⁹⁹ (Fig. 4). Residue R97 in FMDV 3C, corresponding to R84 in PV 3C, clearly has a significant role in the binding of the FMDV 3C protein to the cre/bus RNA (Fig. 7) and is required for VPg uridylylation. Moreover, modification of this residue (to Ser) generated noninfectious RNA transcripts. In addition, we have shown that the FMDV 3C residue R92 (at least in the context of 3C alone) is also important for the uridylylation reaction. RNA transcripts containing a mutation at this position were about 10-fold less infectious than the WT. It should be noted that this residue, which corresponds to R79 in PV, has not been shown to be involved in RNA binding, but it may be significant that the residue is highly conserved among the picornaviruses (except for HAV) (Fig. 4). The correlation between RNA binding activity and the ability to facilitate the uridylylation reaction was further supported by mutagenesis of R95 in FMDV 3C. The R95S mutation had a less significant effect on RNA binding activity than changes to R92 or R97 (Fig. 7) and was also found to have a less severe impact on VPg uridylylation (Fig. 6). However, surprisingly, the R95S mutation resulted in a noninfectious RNA transcript, even though the defect in RNA binding and uridylylation activity caused by this substitution was less severe than that for the R92S mutant, which had proved viable. One explanation for this apparent discrepancy may be that the RNA binding activity of 3C is modulated by the presence of the 3D sequences within 3CD. It is apparent that the RNA binding activity of FMDV 3C alone is not specific for the cre/bus, since it also binds to the S fragment (Fig. 7B), but the residues R92 and R97 are clearly both very important for this function, as modification of these individual residues greatly inhibits binding to both the cre/bus and the S fragment.

It is sometimes difficult to design mutations that have only local effects on protein structure and function. For example, several mutations in the substrate binding site of PV 3C were also found to modify RNA binding (8); conversely, amino acid substitutions in the RNA binding motif of enterovirus 70 3C were observed to modify the proteolytic-processing activity of this enzyme (38). In both cases, it appears that the mutations introduced to probe the function of the protein may have had greater effects on the structure of the protease than anticipated, thus complicating interpretation of the experimental results. In FMDV 3C, the residues R92, R/K95, and R97 are in close proximity on the surface of the FMDV 3C protein (Fig. 5). These residues were targeted for mutagenesis with the idea that the modifications could modify RNA binding but have no effect on the catalytic activities of the protease, since the crystal structure showed that they were fully solvent exposed and did not interact with other amino acids (7). The results obtained validated this hypothesis (Fig. 7 and 8A), since each of the mutants, despite showing major defects in RNA binding activity, was fully functional in proteolytic processing.

Recent studies by Yang et al. (46) indicated that a single point mutation at residue L94 (to Pro), on the C-terminal side of the KFRDI motif on the "back face" of the HRV-14 3C protein, permitted the HRV-14 RNA to replicate using the PV cre. This is clearly a nonconservative modification within the 3C protein and may also be expected to significantly modify the structure of 3C in this region and to influence the nature of the interaction between the cre and the 3C protein. It suggests that there are significant differences in the nature of the interaction between the HRV 3C protein with the HRV cre and the interaction between the same protein and the PV cre. The nature of this interaction remains to be determined at a structural level.

It has become apparent that the RNA polymerase 3D^pol is able to bind directly to VPg (20, 45); initial studies suggested that the VPg binding site is located on the face of the 3D^pol protein opposite from its polymerase active site (9, 47). These observations led to suggestions that multiple 3D^pol molecules are involved in the uridylylation reaction (39), an idea supported by previous data indicating that the enzyme activity of PV 3D^pol displays cooperativity (30). However, it has also been shown that a cleft in HRV 3D^pol is large enough to allow a template-primer duplex to access the active site directly, thus permitting VPg uridylylation (2). Most recently, structural studies by Ferrer-Orta et al. (14) have revealed that the FMDV VPg binds in an elongated conformation to a single 3D^pol molecule so that the Y3 residue of the VPg peptide is precisely positioned for uridylylation by the active site of the same FMDV 3D^pol. Further structural studies will be required to elucidate the details of protein-protein and protein-RNA interactions involved in the uridylylation complex.

Although the isolated peptide VPg can act as a substrate in the uridylylation reaction, it is not clear which form of the molecule is actually modified within infected cells. The VPgpU(pU) could be released by proteolytic processing from a uridylylated precursor. Indeed, VPg may be derived from a number of different precursors, e.g., 3AB or 3BC. It is interesting that the entire PV P3 precursor is required to complement a defect in 3AB, suggesting that the functional precursor for RNA replication is the entire P3 region (42). The presence of multiple forms of 3B (VPg) within FMDV increases the number of possible precursors. Since 3C plays a role in the processing reaction, it seemed logical to assess the abilities of the precursor proteins 3B₃3C and 3B₁₂₃3C to act as substrates in the uridylylation reaction. It has now been shown (Fig. 3) that both of these precursors can act as a substrate for uridylylation, even in the absence of added 3C or 3CD, presumably due to the presence of the 3C sequences within the precursor molecule. Consistent with the enhanced uridylylation of VPg seen with 3CD compared to 3C alone (Fig. 2), it was not surprising to find that the uridylylation of the precursor proteins was still stimulated by the addition of 3CD (Fig. 3). It should be noted that the uridylylation of these precursor proteins appeared much less efficient (over 10-fold) than that of the isolated VPg when they were used at equimolar concentrations; thus, the optimal substrate (if it is distinct from VPg itself) for uridylylation has yet to be determined.

As suggested by our previous studies (29), different RNA transcripts containing the FMDV cre/bus acted as templates for VPg uridylylation with different efficiencies. We have now shown that the presence of the IRES sequence within the transcript, as well as the cre/bus, correlated with optimal activity (Fig. 1). However, it should be noted that the longest transcripts including these elements were not the most efficient (e.g., the FL-RNA) (Fig. 1B). It may be that the presence of additional RNA sequences provided additional binding sites for certain proteins and hence reduced their effective concentrations. Alternatively, the presence of the additional sequences in these naked RNA transcripts may have masked the functional elements to some degree. One possible explanation for these results is that undefined RNA-RNA interactions between sequences located upstream and downstream of the cre/bus affected the template activity of this element. In this context, it is noteworthy that studies by Ramos and Martinez-Salas (36) have demonstrated multiple RNA-RNA interactions between different regions of the FMDV 5' UTR, including the cre/bus. The structure of the minimal HRV-14 cre has recently been determined (40); unexpectedly, the AAACA motif required as the template for VPg uridylylation was not prominently exposed in this structure. It is possible that the presence of other RNA sequences may be required to generate the optimal conformation of this element.

 
We thank A. Arias and E. Domingo for kindly providing a sample of the type C FMDV His-tagged 3D^pol.

A.N. acknowledges a Commonwealth Scholarship and a studentship from the Institute for Animal Health. I.G.G. acknowledges support from the Wellcome Trust. This work was funded in part by grant support from the BBSRC (grant ref. BBS/B/06547) to S.C.

 
* Corresponding author. Mailing address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark. Phone: 45 723 45807. Fax: 45 723 47901. E-mail: grb{at}dfvf.dk.

Present address: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-2280.

Present address: Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5G 1A8.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Andino, R., G. E. Rieckhof, P. L. Achacoso, and D. Baltimore. 1993. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 12:3587-3598.[Medline]
2
2. Appleby, T. C., H. Luecke, J. H. Shim, J. Z. Wu, I. W. Cheney, W. Zhong, L. Vogeley, Z. Hong, and N. Yao. 2005. Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer. J. Virol. 79:277-288.[Abstract/Free Full Text]
3
3. Arias, A., R. Agudo, C. Ferrer-Orta, R. Perez-Luque, A. Airaksinen, E. Brocchi, E. Domingo, N. Verdaguer, and C. Escarmis. 2005. Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding. J. Mol. Biol. 353:1021-1032.[CrossRef][Medline]
4
4. Belsham, G. J., and R. J. Jackson. 2000. Translation initiation on picornavirus RNA, p. 869-900. In N. Sonenberg, J. W. B. Hershey, and M. B. Mathews (ed.), Translational control of gene expression. Monograph 39. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
5
5. Bergmann, E. M., S. C. Mosimann, M. M. Chernaia, B. A. Malcolm, and M. N. James. 1997. The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition. J. Virol. 71:2436-2448.[Abstract]
6
6. Birtley, J. R., and S. Curry. 2005. Crystallization of foot-and-mouth disease virus 3C protease: surface mutagenesis and a novel crystal-optimization strategy. Acta Crystallogr. D 61:646-650.[CrossRef][Medline]
7
7. Birtley, J. R., S. R. Knox, A. M. Jaulent, P. Brick, R. J. Leatherbarrow, and S. Curry. 2005. Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity. J. Biol. Chem. 280:11520-11527.[Abstract/Free Full Text]
8
8. Blair, W. S., J. H. Nguyen, T. B. Parsley, and B. L. Semler. 1996. Mutations in the poliovirus 3CD proteinase S1-specificity pocket affect substrate recognition and RNA binding. Virology 218:1-13.[CrossRef][Medline]
9
9. Boerner, J. E., J. M. Lyle, S. Daijogo, B. L. Semler, S. C. Schultz, K. Kirkegaard, and O. C. Richards. 2005. Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase. J. Virol. 79:7803-7811.[Abstract/Free Full Text]
10
10. Conte, M. R., T. Grune, J. Ghuman, G. Kelly, A. Ladas, S. Matthews, and S. Curry. 2000. Structure of tandem RNA recognition motifs for polypyrimidine tract binding protein reveals novel features of the RRM fold. EMBO J. 19:3132-3141.[CrossRef][Medline]
11
11. Crowder, S., and K. Kirkegaard. 2005. trans-Dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat. Genet. 37:701-709.[CrossRef][Medline]
12
12. Ellard, F. M., J. Drew, W. E. Blakemore, D. I. Stuart, and A. M. King. 1999. Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids. J. Gen. Virol. 80:1911-1918.[Abstract/Free Full Text]
13
13. Falk, M. M., F. Sobrino, and E. Beck. 1992. VPg-gene amplification correlates with infective particle formation in foot-and-mouth-disease virus. J. Virol. 66:2251-2260.[Abstract/Free Full Text]
14
14. Ferrer-Orta, C., A. Arias, R. Agudo, R. Perez-Luque, C. Escarmis, E. Domingo, and N. Verdaguer. 2006. The structure of a protein primer-polymerase complex in the initiation of genome replication. EMBO J. 25:880-888.[CrossRef][Medline]
15
15. Gerber, K., E. Wimmer, and A. V. Paul. 2001. Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J. Virol. 75:10979-10990.[Abstract/Free Full Text]
16
16. Gohara, D. W., C. S. Ha, S. K. B. Ghosh, J. J. Arnold, T. J. Wisniewski, and C. E. Cameron. 1999. Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D^pol) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein Expr. Purif. 17:128-138.[CrossRef][Medline]
17
17. Goodfellow, I. G., Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans. 2000. Identification of a cis-acting replication element within the poliovirus coding region. J. Virol. 74:4590-4600.[Abstract/Free Full Text]
18
18. Goodfellow, I. G., D. Kerrigan, and D. J. Evans. 2003. Structure and function of the poliovirus cis-acting replication element (CRE). RNA 9:124-137.[Abstract/Free Full Text]
19
19. Goodfellow, I. G., C. Polacek, R. Andino, and D. J. Evans. 2003. The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes. J. Gen. Virol. 84:2359-2363.[Abstract/Free Full Text]
20
20. Hope, D. A., S. E. Diamond, and K. Kirkegaard. 1997. Genetic dissection of interaction between poliovirus 3D polymerase and viral protein 3AB. J. Virol. 71:9490-9498.[Abstract]
21
21. King, A. M. Q., D. V. Sangar, T. J. R. Harris, and F. Brown. 1980. Heterogeneity of the genome-linked protein of foot-and-mouth disease virus. J. Virol. 34:627-634.[Abstract/Free Full Text]
22
22. Lobert, P. E., N. Escriou, J. Ruelle, and T. Michiels. 1999. A coding RNA sequence acts as a replication signal in cardioviruses. Proc. Natl. Acad. Sci. USA 96:11560-11565.[Abstract/Free Full Text]
23
23. Mason, P. W., S. V. Bezborodova, and T. M. Henry. 2002. Identification and characterization of a cis-acting replication element (cre) adjacent to the IRES of foot-and-mouth disease virus. J. Virol. 76:9686-9694.[Abstract/Free Full Text]
24
24. McKnight, K. L., and S. M. Lemon. 1996. Capsid coding sequence is required for efficient replication of human rhinovirus-14 RNA. J. Virol. 70:1941-1952.[Abstract]
25
25. McKnight, K. L., and S. M. Lemon. 1998. The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication. RNA 4:1569-1584.[Abstract]
26
26. Morasco, B. J., N. Sharma, J. Parilla, and J. B. Flanegan. 2003. Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis. J. Virol. 77:5136-5144.[Abstract/Free Full Text]
27
27. Mosimann, S. C., M. M. Cherney, S. Sia, S. Plotch, and M. N. James. 1997. Refined X-ray crystallographic structure of the poliovirus 3C gene product. J. Mol. Biol. 273:1032-1047.[CrossRef][Medline]
28
28. Murray, K. E., and D. J. Barton. 2003. Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis. J. Virol. 77:4739-4750.[Abstract/Free Full Text]
29
29. Nayak, A., I. G. Goodfellow, and G. J. Belsham. 2005. Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J. Virol. 79:7698-7706.[Abstract/Free Full Text]
30
30. Pata, J. D., S. C. Shultz, and K. Kirkegaard. 1995. Functional oligomerization of poliovirus RNA dependent RNA polymerase. RNA 1:466-477.[Abstract]
31
31. Pathak, H. B., S. K. B. Ghosh, A. W. Roberts, S. D. Sharma, J. D. Yoder, J. J. Arnold, D. W. Gohara, D. J. Barton, A. V. Paul, and C. E. Cameron. 2002. Structure-function relationships of the RNA-dependent RNA polymerase from poliovirus (3D^pol). J. Biol. Chem. 277:31551-31562.[Abstract/Free Full Text]
32
32. Paul, A. V. 2002. Possible unifying mechanism of picornavirus genome replication, p. 227-246. In B. L. Semler and E. Wimmer (ed.), Molecular biology of picornaviruses. ASM Press, Washington, D.C.
33
33. Paul, A. V., E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer. 2000. Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J. Virol. 74:10359-10370.[Abstract/Free Full Text]
34
34. Paul, A. V., J. Peters, J. Mugavero, J. Yin, J. H. van Boom, and E. Wimmer. 2003. Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus. J. Virol. 77:891-904.[CrossRef][Medline]
35
35. Paul, A. V., J. Yin, J. Mugavero, E. Rieder, Y. Liu, and E. Wimmer. 2003. A "slide-back" mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus. J. Biol. Chem. 278:43951-43960.[Abstract/Free Full Text]
36
36. Ramos, R., and E. Martinez-Salas. 1999. Long-range interactions between structural domains of the aphthovirus internal ribosome entry site (IRES). RNA 5:1374-1383.[Abstract]
37
37. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
38
38. Shih, S. R., C. Chiang, T. C. Chen, C. N. Wu, J. T. Hsu, J. C. Lee, M. J. Hwang, M. L. Li, G. W. Chen, and M. S. Ho. 2004. Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities. J. Biomed. Sci. 11:239-248.[Medline]
39
39. Tellez, A. B., S. Crowder, J. F. Spagnolo, A. A. Thompson, O. B. Peersen, D. L. Brutlag, and K. Kirkegaard. 2006. Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer. J. Mol. Biol. 357:665-675.[CrossRef][Medline]
40
40. Thiviyanathan, V., Y. Yang, K. Kaluarachchi, R. Rijnbrand, D. G. Gorenstein, and S. M. Lemon. 2004. High-resolution structure of a picornavirus internal cis-acting RNA replication element (cre). Proc. Natl. Acad. Sci. USA 101:12688-12693.[Abstract/Free Full Text]
41
41. Tiley, L., A. M. Q. King, and G. J. Belsham. 2003. The foot-and-mouth disease virus cis-acting replication element (cre) can be complemented in trans within infected cells. J. Virol. 77:2243-2246.[Abstract/Free Full Text]
42
42. Towner, J. S., M. M. Mazanet, and B. L. Semler. 1998. Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins. J. Virol. 72:7191-7200.[Abstract/Free Full Text]
43
43. van Ooij, M. J., D. A. Vogt, A. Paul, C. Castro, J. Kuijpers, F. J. van Kuppeveld, C. E. Cameron, E. Wimmer, R. Andino, and W. J. Melchers. 2006. Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative-and positive-strand RNA synthesis. J. Gen. Virol. 87:103-113.[Abstract/Free Full Text]
44
44. Walker, P. A., L. E. Leong, and A. G. Porter. 1995. Sequence and structural determinants of the interaction between the 5'-noncoding region of picornavirus RNA and rhinovirus protease 3C. J. Biol. Chem. 270:14510-14516.[Abstract/Free Full Text]
45
45. Xiang, W., A. Cuconati, D. Hope, K. Kirkegaard, and E. Wimmer. 1998. Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3D^pol with VPg and with genetic variants of 3AB. J. Virol. 72:6732-6741.[Abstract/Free Full Text]
46
46. Yang, Y., R. Rijnbrand, K. L. McKnight, E. Wimmer, A. Paul, A. Martin, and S. M. Lemon. 2002. Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14. J. Virol. 76:7485-7494.[Abstract/Free Full Text]
47
47. Yang, Y., R. Rijnbrand, S. Watowich, and S. M. Lemon. 2004. Genetic evidence for an interaction between a picornaviral cis-acting RNA replication element and 3CD protein. J. Biol. Chem. 279:12659-12667.[Abstract/Free Full Text]
48
48. Yin, J., A. V. Paul, E. Wimmer, and E. Rieder. 2003. Functional dissection of a poliovirus cis-acting replication element [PV-cre(2C)]: analysis of single- and dual-cre viral genomes and proteins that bind specifically to PV-cre RNA. J. Virol. 77:5152-5166.[Abstract/Free Full Text]
49
49. Zell, R., K. Sidigi, E. Bucci, A. Stelzner, and M. Gorlach. 2002. Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C. RNA 8:188-201.[Abstract]

---

Journal of Virology, October 2006, p. 9865-9875, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00561-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Pathak, H. B., Oh, H. S., Goodfellow, I. G., Arnold, J. J., Cameron, C. E. (2008). Picornavirus Genome Replication: ROLES OF PRECURSOR PROTEINS AND RATE-LIMITING STEPS IN oriI-DEPENDENT VPg URIDYLYLATION. J. Biol. Chem. 283: 30677-30688 [Abstract] [Full Text]  
• Amero, C. D., Arnold, J. J., Moustafa, I. M., Cameron, C. E., Foster, M. P. (2008). Identification of the oriI-Binding Site of Poliovirus 3C Protein by Nuclear Magnetic Resonance Spectroscopy. J. Virol. 82: 4363-4370 [Abstract] [Full Text]  
• Shen, M., Wang, Q., Yang, Y., Pathak, H. B., Arnold, J. J., Castro, C., Lemon, S. M., Cameron, C. E. (2007). Human Rhinovirus Type 14 Gain-of-Function Mutants for oriI Utilization Define Residues of 3C(D) and 3Dpol That Contribute to Assembly and Stability of the Picornavirus VPg Uridylylation Complex. J. Virol. 81: 12485-12495 [Abstract] [Full Text]  
• Pathak, H. B., Arnold, J. J., Wiegand, P. N., Hargittai, M. R. S., Cameron, C. E. (2007). Picornavirus Genome Replication: ASSEMBLY AND ORGANIZATION OF THE VPg URIDYLYLATION RIBONUCLEOPROTEIN (INITIATION) COMPLEX. J. Biol. Chem. 282: 16202-16213 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nayak, A. Articles by Belsham, G. J. Search for Related Content PubMed PubMed Citation Articles by Nayak, A. Articles by Belsham, G. J.