scholarly journals Demonstration of Co-Infection and Trans-Encapsidation of Viral RNA In Vitro Using Epitope-Tagged Foot-and-Mouth Disease Viruses

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2433
Author(s):  
Kay Childs ◽  
Nicholas Juleff ◽  
Katy Moffat ◽  
Julian Seago
Keyword(s):  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Flag Epitope ◽  
Experimental Systems ◽  
Infection Dynamics ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
Vp1 Capsid Protein

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies.

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

2020 ◽  
Vol 94 (13) ◽  
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.

scholarly journals 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

2005 ◽  
Vol 79 (12) ◽  
pp. 7698-7706 ◽  
Author(s):  
Arabinda Nayak ◽  
Ian G. Goodfellow ◽  
Graham J. Belsham
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Rna Synthesis ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Coding Region ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Positive Strand Rna

ABSTRACT The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3Dpol. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3Dpol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

scholarly journals Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro

2017 ◽  
Vol 89 (11) ◽  
pp. 2041-2046 ◽  
Author(s):  
Fu-Rong Zhao ◽  
Yin-Li Xie ◽  
Ze-Zhong Liu ◽  
Jun-Jun Shao ◽  
Shi-Fang Li ◽  
...  
Keyword(s):  
Disease Virus ◽  
Lithium Chloride ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Early Stages ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Fmdv Replication

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

2012 ◽  
Vol 12 (1) ◽  
pp. 363-377 ◽  
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

scholarly journals In vitro comparison of foot-and-mouth disease virus subtype variants causing disease in vaccinated cattle

1978 ◽  
Vol 80 (3) ◽  
pp. 451-459 ◽  
Author(s):  
E. C. Anderson ◽  
W. J. Doughty ◽  
J. Anderson ◽  
D. Baber
Keyword(s):  
Disease Virus ◽  
Vaccine Strain ◽  
Complement Fixation ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Radial Immunodiffusion ◽  
Antibody Concentration ◽  
Mouth Disease Virus ◽  
Foot And Mouth

SummaryFoot-and-mouth disease virus isolates of types O, A and SAT 2, from diseased animals in herds routinely vaccinated twice a year were compared antigenically with the vaccine strains in the complement-fixation, neutralization and radial immunodiffusion tests. It was found that strains which had readily infected vaccinated cattle had R values against the vaccine strain in the complement- fixation and radial immunodiffusion tests of 30 or less, while strains causing primary outbreaks with little spread had R values of 30–40. Threefold differences in humoral neutralizing antibody concentration between the field variant and the vaccine strain in sera from vaccinated animals were likely to be significant in terms of protection.

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

2009 ◽  
Vol 29 (3) ◽  
pp. 179-192 ◽  
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

scholarly journals Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

2009 ◽  
Vol 83 (21) ◽  
pp. 11275-11282 ◽  
Author(s):  
Stewart Goodwin ◽  
Tobias J. Tuthill ◽  
Armando Arias ◽  
Richard A. Killington ◽  
David J. Rowlands
Keyword(s):  
Disease Virus ◽  
Receptor Binding ◽  
Virus Assembly ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Precursor Protein ◽  
Capsid Precursor ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACT The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in E scherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin αvβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

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