Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

Clarke, Berwyn; Sangar, D. V.; Burroughs, J. N.; Newton, Susan E; Carroll, Anthony R.; Rowlands, David J.

doi:10.1099/0022-1317-66-12-2615

Cited by 68 publications

(46 citation statements)

References 31 publications

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“…The AcJR4/MR41 recombinants produced two AL-1AB products (lane 11). This may be due to the unusual possession by FMDV of two efficient alternative initiation codons for L (Clarke et al, 1985). Protein 3D and a small amount of the 3CD precursor were also observed.…”

Section: I I :~ĩĩ]~?:[ĩĩĩĩ| ?~!!~:~ĩĩI :~I I ĩĩĩĩĩĩ$I ĩĩI I I I I mentioning

confidence: 97%

Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors

Roosien¹,

Belsham

Ryan

et al. 1990

Journal of General Virology

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Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor P1-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins lAB, 1C and 1D were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike P1 processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P 1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting Lcontaining cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-P1 junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.

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Section: I I :~ĩĩ]~?:[ĩĩĩĩ| ?~!!~:~ĩĩI :~I I ĩĩĩĩĩĩ$I ĩĩI I I I I mentioning

confidence: 97%

Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors

Roosien¹,

Belsham

Ryan

et al. 1990

Journal of General Virology

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“…The upstream initiation codon in FMDV RNA is recognized by internal initiation, mediated by the internal ribosomal entry site (IRES); recognition of the downstream initiation codon is most likely via the leaky scanning method (Kozak, 1991) by ribosomes that skipped the upstream initiation codon (Belsham, 1992). Initiation of FMDV RNA translation at the two initiation codons, designated AUGL, b and AUGLb (Clarke et al, 1985), results in two polyproteins that are cleaved to yield two proteases, p20 (or Lab) and p16 (or Lb), with identical carboxy termini. The p16 protein is post-translationally modified (Sangar et aL, 1987).…”

Section: Introductionmentioning

confidence: 99%

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Thomas

Rijnbrand

Voorma

1996

Journal of General Virology

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Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGL~b) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGL~ b for wildtype and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of elF-2 was measured over a broad range of mRNA concentrations. In conclusion, elF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

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“…A conserved feature among the FMDV serotypes is that translation of the polyprotein is initiated at two different AUG codons, one at the 3' end of the IRES and another located 84 nt downstream (1,5). This results in the production of two forms of the leader (L) proteinase, Lab and Lb, in infected cells (28).…”

Section: Equine Rhinitis a Virus (Erav) Has Recently Been Classified mentioning

confidence: 99%

“…In this model, translation is initiated 12 to 15 nucleotides (nt), downstream of a polypyrimidine tract in a location that is immediately 3' to the core structural elements that define the IRES (2,3,23). A type III IRES is found in hepatoviruses, and although this model is much less studied, it appears to share features of both types I and II (4).A conserved feature among the FMDV serotypes is that translation of the polyprotein is initiated at two different AUG codons, one at the 3' end of the IRES and another located 84 nt downstream (1,5). This results in the production of two forms of the leader (L) proteinase, Lab and Lb, in infected cells (28).…”

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confidence: 99%

Internal Ribosomal Entry Site-Mediated Translation Initiation in Equine Rhinitis A Virus: Similarities to and Differences from That of Foot-and-Mouth Disease Virus

Hinton

Feng

Crabb

2000

J Virol

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Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinasesLab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.Equine rhinitis A virus (ERAV), formerly known as Equine rhinovirus 1, is a member of the Picornaviridae family and has been recently reclassified as the only non-foot-and-mouth disease virus (non-FMDV) member of the genus Aphthovirus (26). This reclassification was based largely on nucleotide sequence determination of the ERAV genome (14, 32), although it is also consistent with many of the known physicochemical and biological properties of the virus (20). ERAV infection of horses results in an acute febrile respiratory disease that is accompanied by viremia and persistent virus shedding in the urine and feces (see reference 30 for a review). It is also pathogenic for a broad range of other animal species, including humans (24, 25). The recent finding that strains of ERAV which are noncytopathic in in vitro-cultured cells are responsible for outbreaks of febrile respiratory disease in horses suggests that the virus has been underdiagnosed and that its relative significance as an equine pathogen may have been underestimated (15). Further investigation into the epidemiology and pathogenesis of this virus is clearly required. In this paper we characterize the role of the ERAV 5' nontranslated region (5'-NTR) in translation initiation, an important pathogenic determinant in picornaviruses.Picornaviruses initiate translation in a cap-inde...

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Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

Cited by 68 publications

References 31 publications

Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors

Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Internal Ribosomal Entry Site-Mediated Translation Initiation in Equine Rhinitis A Virus: Similarities to and Differences from That of Foot-and-Mouth Disease Virus

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