BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

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(F)uridylylated Peptides Linked to VPg1 of Foot-and- Mouth Disease Virus (FMDV): Design, Synthesis and X-Ray Crystallography of the Complexes with FMDV RNA-Dependent RNA Polymerase

Molecules ◽

10.3390/molecules24132360 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2360 ◽

Cited By ~ 2

Author(s):

Sonia de Castro ◽

Cristina Ferrer-Orta ◽

Alberto Mills ◽

Gloria Fernández-Cureses ◽

Federico Gago ◽

...

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Viral Rna ◽

Hydroxyl Group ◽

Rna Dependent Rna Polymerase ◽

X Ray ◽

Mouth Disease Virus ◽

Foot And Mouth

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5′-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the β9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.

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Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

Journal of General Virology ◽

10.1099/vir.0.067751-0 ◽

2014 ◽

Vol 95 (12) ◽

pp. 2649-2657 ◽

Cited By ~ 9

Author(s):

Sophie Forrest ◽

Zoe Lear ◽

Morgan R. Herod ◽

Martin Ryan ◽

David J. Rowlands ◽

...

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Rna Aptamers ◽

Gfp Expression ◽

Subgenomic Replicon ◽

Replication Complex ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Dose Dependent

We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2′-C-methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.

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Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA

Acta Virologica ◽

10.4149/av_2016_01_85 ◽

2016 ◽

Vol 60 (01) ◽

pp. 85-93 ◽

Cited By ~ 3

Author(s):

H.-M. Wang ◽

X.-Z. Xia ◽

G.-X. HU ◽

L. Yu ◽

H.-B. He

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Viral Rna ◽

Mouth Disease Virus ◽

Foot And Mouth

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The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

10.1101/2020.01.10.901801 ◽

2020 ◽

Author(s):

Joseph C. Ward ◽

Lidia Lasecka-Dykes ◽

Chris Neil ◽

Oluwapelumi Adeyemi ◽

Sarah Gold ◽

...

Keyword(s):

Disease Virus ◽

Infectious Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Genome Replication ◽

Entry Site ◽

Stem Loop ◽

Initiation Of Translation ◽

Mouth Disease Virus ◽

Foot And Mouth

AbstractThe positive stranded RNA genomes of picornaviruses comprise a single large open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). Foot-and-mouth disease virus (FMDV) has an unusually large 5′ UTR (1.3 kb) containing five structural domains. These include the internal ribosome entry site (IRES), which facilitates initiation of translation, and the cis-acting replication element (cre). Less well characterised structures are a 5′ terminal 360 nucleotide stem-loop, a variable length poly-C-tract of approximately 100-200 nucleotides and a series of two to four tandemly repeated pseudoknots (PKs). We investigated the structures of the PKs by selective 2′ hydroxyl acetylation analysed by primer extension (SHAPE) analysis and determined their contribution to genome replication by mutation and deletion experiments. SHAPE and mutation experiments confirmed the importance of the previously predicted PK structures for their function. Deletion experiments showed that although PKs are not essential for replication, they provide genomes with a competitive advantage. However, although replicons and full-length genomes lacking all PKs were replication competent, no infectious virus was rescued from genomes containing less than one PK copy. This is consistent with our earlier report describing the presence of putative packaging signals in the PK region.

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Dynamics of picornavirus RNA replication within infected cells

Journal of General Virology ◽

10.1099/vir.0.83385-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 485-493 ◽

Cited By ~ 18

Author(s):

Graham J. Belsham ◽

Preben Normann

Keyword(s):

Disease Virus ◽

Rna Synthesis ◽

Foot And Mouth Disease ◽

Rna Replication ◽

Mouth Disease ◽

Swine Vesicular Disease Virus ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells ◽

Vesicular Disease

Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks the initiation of negative-strand synthesis. We have now examined the dynamics of RNA replication, measured by quantitative RT-PCR, within cells infected with either swine vesicular disease virus (an enterovirus) or foot-and-mouth disease virus as regulated by the presence or absence of guanidine. Following the removal of guanidine from the infected cells, RNA replication occurs after a significant lag phase. This restoration of RNA synthesis requires de novo protein synthesis. Viral RNA can be maintained for at least 72 h within cells in the absence of apparent replication but guanidine-resistant virus can become predominant. Amino acid substitutions within the 2C protein that confer guanidine resistance to swine vesicular disease virus and foot-and-mouth disease virus have been identified. Even when RNA synthesis is well established, the addition of guanidine has a major impact on the level of RNA replication. Thus, the guanidine-sensitive step in RNA synthesis is important throughout the virus life cycle in cells.

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Extent of reduction of foot-and-mouth disease virus RNA load in oesophageal–pharyngeal fluid after peak levels may be a critical determinant of virus persistence in infected cattle

Journal of General Virology ◽

10.1099/vir.0.19538-0 ◽

2004 ◽

Vol 85 (2) ◽

pp. 415-421 ◽

Cited By ~ 15

Author(s):

Zhidong Zhang ◽

Ciara Murphy ◽

Melvyn Quan ◽

Jeanette Knight ◽

Soren Alexandersen

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Viral Rna ◽

Critical Determinant ◽

Virus Persistence ◽

Virus Clearance ◽

Virus Rna ◽

Mouth Disease Virus ◽

Foot And Mouth

To investigate whether foot-and-mouth disease virus (FMDV) RNA loads in oesophageal–pharyngeal fluid (OP-fluid) in the early course of infection is related to the outcome of virus persistence, viral RNA in OP-fluid samples from cattle experimentally infected with FMDV type O was quantitatively analysed by using a quantitative real-time RT-PCR. Viral RNA was detected within 24 h post-infection (p.i.) in all infected animals. Rapid virus replication led to peak levels of viral RNA load by 30–53 h p.i., and then the load declined at various rates. In some animals (n=12, so-called non-carriers) viral RNA became undetectable between 7 and 18 days p.i. In contrast, in persistently infected animals (n=12, so-called carriers) viral RNA persisted in OP-fluid samples at detectable levels beyond 28 days p.i. Analysis of early viral decay/clearance and virus clearance half-life in OP-fluid samples showed that the extent of reduction of viral RNA in OP-fluid samples immediately following peak levels is a critical determinant of the outcome of FMDV persistence.

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Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region

10.1101/2021.01.04.425359 ◽

2021 ◽

Author(s):

Lidia Lasecka-Dykes ◽

Fiona Tulloch ◽

Peter Simmonds ◽

Garry A. Luke ◽

Paolo Ribeca ◽

...

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Viral Rna ◽

Rna Structures ◽

Protein Coding ◽

Rna Translation ◽

Mouth Disease Virus ◽

Wide Range ◽

Foot And Mouth

ABSTRACTRNA structure plays a crucial role in the replication of positive sense RNA viruses and can form functional elements within the untranslated regions (UTRs) and the protein coding sequences (or open reading frames (ORFs)). While RNA structures in the UTRs of several picornaviruses have been functionally characterised, the roles of putative RNA structures predicted for the ORF remain largely undefined. Here we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome and predicted the existence of 53 evolutionarily conserved RNA structures within the ORF. Forty-five (45) of these structures were located in the regions encoding the non-structural proteins (nsps). To investigate if the structures in the regions encoding the nsps are required for FMDV replication we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimise RNA secondary structures while preserving protein coding, native dinucleotide frequencies and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV sub-genomic replicon. We found that three of the RNA structures, all at the 3’ termini of the FMDV ORF, were critical for replicon replication. Contrastingly, disruption of the other 42 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains.IMPORTANCESome RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 45 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the non-structural proteins, only three are essential for replication of an FMDV sub-genomic replicon. Replicons replication is only dependent on their RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify if the remaining 42 RNA structures have other roles in virus replication or transmission, they may provide ideal targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

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