The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus of the Picornaviridae. Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures where virus genome replication takes place. In this report, using fluorescence and electron microscopy, membrane rearrangements in the cytoplasm of FMDV-infected BHK-38 cells are documented. At 1·5–2·0 h post-infection, free ribosomes, fragmented rough endoplasmic reticulum, Golgi and smooth membrane-bound vesicles accumulated on one side of the nucleus. Newly synthesized viral RNA was localized to this region of the cell. The changes seen in FMDV-infected cells distinguish this virus from other members of the Picornaviridae, such as poliovirus. Firstly, the collapse of cellular organelles to one side of the cell has not previously been observed for other picornaviruses. Secondly, the membrane vesicles, induced by FMDV, appear distinct from those induced by other picornaviruses such as poliovirus and echovirus 11 since they are relatively few in number and do not aggregate into densely packed clusters. Additionally, the proportion of vesicles with double membranes is considerably lower in FMDV-infected cells. These differences did not result from the use of BHK-38 cells in this study, as infection of these cells by another picornavirus, bovine enterovirus (a close relative of poliovirus), resulted in morphological changes similar to those reported for poliovirus-infected cells. With conventional fixation, FMDV particles were not seen; however, following high-pressure freezing and freeze-substitution, many clusters of virus-like particles were seen.

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

Journal of Virology ◽

10.1128/jvi.00469-16 ◽

2016 ◽

Vol 90 (15) ◽

pp. 6864-6883 ◽

Cited By ~ 6

Author(s):

Morgan R. Herod ◽

Cristina Ferrer-Orta ◽

Eleni-Anna Loundras ◽

Joseph C. Ward ◽

Nuria Verdaguer ◽

...

Keyword(s):

Disease Virus ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Rna Replication ◽

Mouth Disease ◽

Viral Rna ◽

Genome Replication ◽

Replication Complex ◽

Mouth Disease Virus ◽

Foot And Mouth

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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Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.00341-20 ◽

2020 ◽

Vol 94 (13) ◽

Cited By ~ 4

Author(s):

Gisselle N. Medina ◽

Paul Azzinaro ◽

Elizabeth Ramirez-Medina ◽

Joseph Gutkoska ◽

Ying Fang ◽

...

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Type I ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells ◽

Viral Attenuation

ABSTRACT Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.

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Quantitative Proteomics by Amino Acid Labeling in Foot-and-Mouth Disease Virus (FMDV)-Infected Cells

Journal of Proteome Research ◽

10.1021/pr300611e ◽

2012 ◽

Vol 12 (1) ◽

pp. 363-377 ◽

Cited By ~ 17

Author(s):

Yu Ye ◽

Guangrong Yan ◽

Yongwen Luo ◽

Tiezhu Tong ◽

Xiangtao Liu ◽

...

Keyword(s):

Amino Acid ◽

Disease Virus ◽

Quantitative Proteomics ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells ◽

Amino Acid Labeling

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Activation of Porcine Natural Killer Cells and Lysis of Foot-and-Mouth Disease Virus Infected Cells

Journal of Interferon & Cytokine Research ◽

10.1089/jir.2008.0058 ◽

2009 ◽

Vol 29 (3) ◽

pp. 179-192 ◽

Cited By ~ 20

Author(s):

Felix N. Toka ◽

Charles K. Nfon ◽

Harry Dawson ◽

D. Mark Estes ◽

William T. Golde

Keyword(s):

Natural Killer Cells ◽

Natural Killer ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Killer Cells ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells

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Presentation of antigenic sites from foot-and-mouth disease virus on the surface of baculovirus and in the membrane of infected cells

Archives of Virology ◽

10.1007/s007050070058 ◽

2000 ◽

Vol 145 (9) ◽

pp. 1815-1828 ◽

Cited By ~ 22

Author(s):

C. Tami ◽

M. Farber ◽

E. L. Palma ◽

O. Taboga

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Antigenic Sites ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells

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Use of in Situ Hybridization for the Detection of Foot-and-Mouth Disease Virus in Cell Culture

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100409 ◽

1989 ◽

Vol 1 (4) ◽

pp. 329-332 ◽

Cited By ~ 3

Author(s):

Richard F. Meyer ◽

Corrie C. Brown ◽

Thomas W. Molitor ◽

Vikram N. Vakharia

Keyword(s):

Disease Virus ◽

Detection System ◽

Cdna Probe ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Rna Probes ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at lo-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.

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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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The Foot-and-Mouth Disease Virus cis-Acting Replication Element (cre) Can Be Complemented in trans within Infected Cells

Journal of Virology ◽

10.1128/jvi.77.3.2243-2246.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2243-2246 ◽

Cited By ~ 16

Author(s):

Laurence Tiley ◽

Andrew M. Q. King ◽

Graham J. Belsham

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Temperature Sensitive ◽

Stem Loop ◽

Cis Acting ◽

Stem Loop Structure ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Infected Cells

ABSTRACT A temperature-sensitive (ts) mutation was identified within the 5′-untranslated region of foot-and-mouth disease virus (FMDV) RNA. The mutation destabilizes a stem-loop structure recently identified as a cis-acting replication element (cre). Genetic analyses indicated that the ts defect in virus replication could be complemented. Thus, the FMDV cre can function in trans. It is suggested that the cre be renamed a 3B-uridylylation site (bus).

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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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