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1.
J Virol ; 98(5): e0035024, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38591900

RESUMO

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.


Assuntos
Calicivirus Felino , Proteínas do Capsídeo , Endossomos , RNA Viral , Animais , Gatos , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/metabolismo , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Calicivirus Felino/fisiologia , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Endossomos/virologia , Endossomos/metabolismo , Genoma Viral , Lipossomos/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Liberação de Vírus
2.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581104

RESUMO

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region. The regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III, and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition was antagonized by the viral 3C protease through the cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K1-364, retained partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product, hnRNP K364-465, became a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and the modulation of ITAFs, providing new insights into translational control during viral infection.IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving the proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1-364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus and provide new insights into translational control during viral infection.


Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Febre Aftosa/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Sítios Internos de Entrada Ribossomal/fisiologia , Transativadores/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Proteases Virais 3C , Animais , Linhagem Celular , Cricetinae , Vírus da Febre Aftosa/genética , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas , RNA Mensageiro , Proteínas Virais/genética
3.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161169

RESUMO

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3Dpol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3Dpol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.


Assuntos
Regiões 5' não Traduzidas , Vírus da Febre Aftosa/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Sítios Internos de Entrada Ribossomal/fisiologia , RNA Viral/biossíntese , Replicação Viral/fisiologia , Animais , Linhagem Celular , Vírus da Febre Aftosa/genética , Regulação Viral da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunoprecipitação , Ligação Proteica , RNA Viral/genética , Transcriptoma
4.
J Virol ; 95(1)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33028719

RESUMO

The low fidelity of foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase allows FMDV to exhibit high genetic diversity. Previously, we showed that the genetic diversity of FMDV plays an important role in virulence in suckling mice. Here, we mutated the amino acid residue Phe257, located in the finger domain of FMDV polymerase and conserved across FMDV serotypes, to a cysteine (F257C) to study the relationship between viral genetic diversity, virulence, and transmissibility in natural hosts. The single amino acid substitution in FMDV polymerase resulted in a high-fidelity virus variant, rF257C, with growth kinetics indistinguishable from those of wild-type (WT) virus in cell culture, but it displayed smaller plaques and impaired fitness in direct competition assays. Furthermore, we found that rF257C was attenuated in vivo in both suckling mice and pigs (one of its natural hosts). Importantly, contact exposure experiments showed that the rF257C virus exhibited reduced transmissibility compared to that of wild-type FMDV in the porcine model. This study provides evidence that FMDV genetic diversity is important for viral virulence and transmissibility in susceptible animals. Given that type O FMDV exhibits the highest genetic diversity among all seven serotypes of FMDV, we propose that the lower polymerase fidelity of the type O FMDV could contribute to its dominance worldwide.IMPORTANCE Among the seven serotypes of FMDV, serotype O FMDV have the broadest distribution worldwide, which could be due to their high virulence and transmissibility induced by high genetic diversity. In this paper, we generated a single amino acid substitution FMDV variant with a high-fidelity polymerase associated with viral fitness, virulence, and transmissibility in a natural host. The results highlight that maintenance of viral population diversity is essential for interhost viral spread. This study provides evidence that higher genetic diversity of type O FMDV could increase both virulence and transmissibility, thus leading to their dominance in the global epidemic.


Assuntos
Vírus da Febre Aftosa/patogenicidade , Febre Aftosa/virologia , RNA Polimerase Dependente de RNA/fisiologia , Proteínas não Estruturais Virais/fisiologia , Animais , Linhagem Celular , Cricetinae , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/genética , Aptidão Genética , Variação Genética , Camundongos , Mutação , Fenótipo , RNA Polimerase Dependente de RNA/genética , Suínos , Proteínas não Estruturais Virais/genética , Virulência
5.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32493820

RESUMO

Foot-and-mouth disease (FMD), which is caused by FMD virus (FMDV), remains a major plague among cloven-hoofed animals worldwide, and its outbreak often has disastrous socioeconomic consequences. A live-attenuated FMDV vaccine will greatly facilitate the global control and eradication of FMD, but a safe and effective attenuated FMDV vaccine has not yet been successfully developed. Here, we found that the internal ribosome entry site (IRES) element in the viral genome is a critical virulence determinant of FMDV, and a nucleotide substitution of cytosine (C) for guanine (G) at position 351 of the IRES endows FMDV with temperature-sensitive and attenuation (ts&att) phenotypes. Furthermore, we demonstrated that the C351G mutation of IRES causes a temperature-dependent translation defect by impairing its binding to cellular pyrimidine tract-binding protein (PTB), resulting in the ts&att phenotypes of FMDV. Natural hosts inoculated with viruses carrying the IRES C351G mutation showed no clinical signs, viremia, virus excretion, or viral transmission but still produced a potent neutralizing antibody response that provided complete protection. Importantly, the IRES C351G mutation is a universal determinant of the ts&att phenotypes of different FMDV strains, and the C351G mutant was incapable of reversion to virulence during in vitro and in vivo passages. Collectively, our findings suggested that manipulation of the IRES, especially its C351G mutation, may serve as a feasible strategy to develop live-attenuated FMDV vaccines.IMPORTANCE The World Organization for Animal Health has called for global control and eradication of foot-and-mouth disease (FMD), the most economically and socially devastating disease affecting animal husbandry worldwide. Live-attenuated vaccines are considered the most effective strategy for prevention, control, and eradication of infectious diseases due to their capacity to induce potent and long-lasting protective immunity. However, efforts to develop FMD virus (FMDV) live-attenuated vaccines have achieved only limited success. Here, by structure-function study of the FMDV internal ribosome entry site (IRES), we find that the C351 mutation of the IRES confers FMDV with an ideal temperature-sensitive attenuation phenotype by decreasing its interaction with cellular pyrimidine tract-binding protein (PTB) to cause IRES-mediated temperature-dependent translation defects. The temperature-sensitive attenuated strains generated by manipulation of the IRES address the challenges of FMDV attenuation differences among various livestock species and immunogenicity maintenance encountered previously, and this strategy can be applied to other viruses with an IRES to rationally design and develop live-attenuated vaccines.


Assuntos
Vírus da Febre Aftosa/genética , Sítios Internos de Entrada Ribossomal/genética , Animais , Anticorpos Neutralizantes/metabolismo , Bovinos , Feminino , Febre Aftosa/virologia , Vírus da Febre Aftosa/metabolismo , Vírus da Febre Aftosa/patogenicidade , Regulação Viral da Expressão Gênica/genética , Sítios Internos de Entrada Ribossomal/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Ribossomos/genética , Suínos , Vacinas Atenuadas , Virulência/genética , Replicação Viral/genética
6.
J Gen Virol ; 100(2): 187-198, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30547855

RESUMO

In this study, ten sites on the N terminus and different surface variable regions (VRs) of the bovine parvovirus (BPV) VP2 capsid protein were selected according to an alignment of its sequence with that of the BPV-1 strain HADEN for insertion of the type O foot-and-mouth disease virus (FMDV) conserved neutralizing epitope 8E8. Ten epitope-chimeric BPV VP2 capsid proteins carrying the 8E8 epitope were expressed in Sf9 cells, and electron micrographs demonstrated that these fusion proteins self-assembled into virus-like particles (VLPs) with properties similar to those of natural BPV virions. Immunofluorescence assay (IFA) and Western blot analysis demonstrated that each of the ten epitope-chimeric VLPs reacted with both anti-BPV serum and anti-type O FMDV mAb 8E8. These results indicated that insertions of the 8E8 epitope at these sites on the BPV VP2 protein did not interfere with the immunoreactivity of VP2 or VLP formation, and that the exogenous epitope 8E8 was correctly expressed in BPV VLPs. In addition, anti-BPV IgG antibodies were induced in mice by intramuscular inoculation with each of the ten chimeric VLPs, indicating that the immunogenicity of the chimeric VLPs was not disrupted. Importantly, potent anti-FMDV viral neutralizing (VN) antibodies, which exhibited the highest titre of 1 : 176, were induced by two chimeric VLPs, rBPV-VLP-8E8(391) and rBPV-VLP-8E8(395), in which the 8E8 epitope was inserted into positions 391/392 and 395/396, respectively, in the VR VIII of BPV VP2. Our results demonstrated that the 391/392 and 395/396 positions in the VR VIII of the BPV VP2 protein can effectively display a foreign epitope, making this an attractive approach for the design of nanoparticle-vectored and epitope-based vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Bocavirus/genética , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Proteínas do Capsídeo/genética , Portadores de Fármacos , Epitopos/genética , Vírus da Febre Aftosa/genética , Imunoglobulina G/sangue , Injeções Intramusculares , Camundongos , Células Sf9 , Spodoptera , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética
7.
Arch Virol ; 162(12): 3875-3880, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28884236

RESUMO

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8TLLEDRILT16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr8 and Asp12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.


Assuntos
Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Sorogrupo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Western Blotting , Bovinos , Doenças dos Bovinos/imunologia , Sequência Conservada , Análise Mutacional de DNA , Mapeamento de Epitopos , Febre Aftosa/imunologia , Mutagênese Sítio-Dirigida , Ligação Proteica , Alinhamento de Sequência
8.
Arch Virol ; 162(7): 1877-1885, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28258408

RESUMO

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, a potent FMDV serotype-independent monoclonal antibody (MAb) 3D9 was generated. Screening of a phage-displayed random 12-peptide library revealed that MAb 3D9 bound to phages displaying a consensus motif GVYxxAYxW that is highly homologous to the 89GVYxxxxxxxAYxxxxW105 motif in the FMDV VP2 protein. Importantly, this conformational epitope was highly conserved among all seven serotypes of FMDV analyzed in sequence alignments. To further verify the authentic epitope recognized by MAb 3D9, a FMDV O/YS/CHA/05 mutant virus V90A was generated using a reverse genetics system. The results revealed that Val90 was an important residue for MAb 3D9 binding within this conformational epitope. Thus, we finely mapped a conserved conformational epitope onto the FMDV VP2 protein. These results could be applied in the development of epitope-based vaccines and suitable MAb-based diagnostic methods for various FMDV serotype-independent tests.


Assuntos
Proteínas do Capsídeo/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Sequência Conservada , Cricetinae , Epitopos , Camundongos
9.
Virus Genes ; 53(6): 840-847, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28634750

RESUMO

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.


Assuntos
Vírus da Febre Aftosa/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Replicação Viral/genética , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Cricetinae , Replicação do DNA/genética , Febre Aftosa/virologia , Especificidade de Hospedeiro/genética , Deleção de Sequência/genética , Suínos , Virulência/genética
10.
J Gen Virol ; 97(4): 901-911, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26795299

RESUMO

The 5' untranslated region (5'UTR) of foot-and-mouth disease virus (FMDV) contains an internal ribosome entry site (IRES) that facilitates translation initiation of the viral ORF in a 5' (m7GpppN) cap-independent manner. IRES elements are responsible for the virulence phenotypes of several enteroviruses. Here, we constructed a chimeric virus in which the IRES of FMDV was completely replaced with that of bovine rhinitis B virus (BRBV) in an infectious clone of serotype O FMDV. The resulting IRES-replaced virus, FMDV(BRBV), replicated as efficiently as WT FMDV in hamster-derived BHK-21 cells, but was restricted for growth in porcine-derived IBRS-2, PK-15 and SK-6 cells, which are susceptible to WT FMDV. To identify the genetic determinants of FMDV underlying this altered cell tropism, a series of IRES-chimeric viruses were constructed in which each domain of the FMDV IRES was replaced with its counterpart from the BRBV IRES. The replication kinetics of these chimeric viruses in different cell lines revealed that the growth restriction phenotype in porcine-derived cells was produced after the replacement of domain 3 or 4 in the FMDV IRES. Furthermore, the change in FMDV cell tropism due to IRES replacement in porcine-derived cells was mainly attributed to a decline in cell-specific IRES translation initiation efficiency. These findings demonstrate that IRES domains 3 and 4 of FMDV are novel cell-specific cis-elements for viral replication in vitro and suggest that IRES-mediated translation determines the species specificity of FMDV infection in vivo.


Assuntos
Vírus da Febre Aftosa/genética , Regulação Viral da Expressão Gênica , Herpesvirus Bovino 1/genética , Sítios Internos de Entrada Ribossomal , Vírus Reordenados/genética , Tropismo Viral , Animais , Sequência de Bases , Linhagem Celular , Quimera/genética , Cricetulus , Células Epiteliais/patologia , Células Epiteliais/virologia , Vírus da Febre Aftosa/crescimento & desenvolvimento , Especificidade de Hospedeiro , Rim/patologia , Rim/virologia , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , RNA Viral/genética , RNA Viral/metabolismo , Vírus Reordenados/crescimento & desenvolvimento , Replicon , Ribossomos , Suínos , Replicação Viral
11.
Arch Virol ; 161(2): 365-75, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26563318

RESUMO

Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae that has caused severe economic losses in many countries of the world. Regular vaccinations have been effectively used to control foot-and-mouth disease (FMD) in countries where the disease is enzootic. Distinguishing between infected and vaccinated animals in herds after immunization is an important component of effective eradication strategies. Nonstructural protein (NSP) 3B of FMDV is part of a larger antigen that is used for this differential diagnosis. In this study, an FMDV serotype-independent monoclonal antibody (MAb) against NSP 3B, 5D12, was generated. Using western blot, it was revealed that MAb 5D12 binds to three fragments of 3B displaying the motifs G(1)PYAGPLERQKPLK(14), K(18)LPQQEGPYAGPMER(32) and V(45)KEGPYEGPVKKPVA(59). The motif G(1)PYAGPLERQKPLK(14) was chosen for further mapping. Different truncated motifs derived from the motif G(1)PYAGPLERQKPLK(14) were expressed as GST-fusion constructs for western blot analysis. The results showed that the 5-aa peptide P(2)YAGP(6) was the minimal epitope reactive to MAb 5D12. Subsequent alanine-scanning mutagenesis analysis revealed that Pro(2), Gly(5) and Pro(6) were crucial for MAb 5D12 binding to P(2)YAGP(6). Furthermore, through sequence alignment analysis, the epitope PxxGP recognized by 5D12 was found to be present not only in 3B-1 but also in 3B2 and 3B3 and was highly conserved in seven serotypes of FMDV strains. Western blot analysis also revealed that the peptide epitope could be recognized by sera from FMDV-infected pigs and cattle. Thus, the 5D12-recognized 3B epitope identified here provides theoretical support for the development of MAb 5D12 as a differential diagnosis reagent for FMDV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Linhagem Celular , Sequência Conservada , Cricetinae , Análise Mutacional de DNA , Mapeamento de Epitopos , Epitopos/genética , Vírus da Febre Aftosa/genética , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas não Estruturais Virais/genética
12.
Arch Virol ; 161(10): 2705-16, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27422396

RESUMO

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, a series of outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope have the potential to provide protective immunity against diverse subtypes of FMDV serotype A and to protect against future pandemics. In this study, we produced an A serotype FMDV-specific monoclonal antibody (MAb) against the viral capsid protein VP1, designated 9A9, that potently neutralized FMDV A/JLYS/CHA/2014 with a 50 % neutralization titer (NT50) of 4,096. GST-fusion proteins expressing truncated peptides of VP1 were subjected to Western blot analysis using MAb 9A9, and it was found that the peptide (143)RGDLGPLAARL(153) of VP1 was the minimal epitope for MAb 9A9 binding. Western blot analysis also revealed that the epitope peptide could be recognized by positive sera from serotype A FMDV-infected pigs and cattle. Subsequent alanine-scanning mutagenesis analysis revealed that residues Gly(147) and Leu(149) of the 9A9-recognized epitope are crucial for MAb 9A9 binding. Furthermore, under immunological pressure selected by MAb 9A9, a single amino acid residue replacement (L149P) occurred in a viral neutralization-escape mutant, which verified the location of a critical residue of this epitope at Leu(149). Importantly, the epitope (143)RGDLGPLAARL(153) was highly conserved among different topotypes of serotype A FMDV strains in sequence alignment analysis. Thus, the results of this study could have application potential in the development of epitope-based vaccines and a suitable MAb-based diagnostic method for detection of type A FMDV as well as quantitation of antibodies against FMDV serotype A.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Vírus da Febre Aftosa/imunologia , Sorogrupo , Animais , Proteínas do Capsídeo/genética , Análise Mutacional de DNA , Epitopos de Linfócito B/genética , Evasão da Resposta Imune , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Testes de Neutralização
13.
J Virol ; 88(8): 4008-20, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24453363

RESUMO

UNLABELLED: Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued D5N, A38V, and DAMM mutants but not the M194I and M296V mutants are high-fidelity RdRp variants. Mutagen resistance assays revealed that the higher replication fidelity was associated with higher-level resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains. IMPORTANCE: The ribavirin-isolated poliovirus (PV) RdRp G64S variant, the polymerases of which were of high replication fidelity, was attenuated in vivo. It has been proposed (M. Vignuzzi, E. Wendt, and R. Andino, Nat. Med. 14:154-161, http://dx.doi.org/10.1038/nm1726) that modulation of replication fidelity is a promising approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high-fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via several attenuated FMDV high-fidelity RdRp variants. Our findings may aid in the rational design of a new type of vaccine strain.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/patogenicidade , Febre Aftosa/virologia , Variação Genética , Ribavirina/farmacologia , Animais , Sequência de Bases , Feminino , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação de Sentido Incorreto , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência/efeitos dos fármacos
14.
Arch Virol ; 159(10): 2641-50, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24888311

RESUMO

Previous studies have shown that RNA viruses can be attenuated by either increased or decreased viral polymerase replication fidelity. Although foot-and-mouth disease virus (FMDV) high-fidelity RNA-dependent RNA polymerase (RdRp) variants with an attenuated phenotype have been isolated using mutagens, no FMDV mutant with a low-fidelity polymerase has been documented to date. Here, we describe the generation of several FMDV RdRp mutants using site-directed mutagenesis via a reverse genetic system. Mutation frequency assays confirmed that five rescued FMDV RdRp mutant populations had lower replication fidelity than the wild-type virus population, which allowed us to assess the effects of the change in replication fidelity on the virus phenotype. These low-fidelity FMDV RdRp mutants showed increased sensitivity to ribavirin or 5-fluorouracil (5-FU) treatment without a loss of growth capacity in cell cultures. In addition, decreased fitness and attenuated virulence were observed for the RdRp mutants with lower fidelity. Importantly, based on a quantitative analysis for fidelity and virulence, we concluded that lower replication fidelity is associated with a more attenuated virus phenotype. These results further contribute to our understanding of the replication fidelity of polymerases of RNA viruses and its relationship to virulence attenuation.


Assuntos
Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/genética , RNA Polimerase Dependente de RNA/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Cricetinae , Farmacorresistência Viral/genética , Fluoruracila/farmacologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Taxa de Mutação , RNA Viral/biossíntese , RNA Viral/genética , Ribavirina/farmacologia , Análise de Sequência de RNA
15.
Arch Virol ; 159(4): 657-67, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24122111

RESUMO

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.


Assuntos
Ácidos/toxicidade , Farmacorresistência Viral , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/genética , Viabilidade Microbiana/efeitos dos fármacos , Seleção Genética , Substituição de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Cricetinae , Análise Mutacional de DNA , Vírus da Febre Aftosa/isolamento & purificação , Vírus da Febre Aftosa/fisiologia , Concentração de Íons de Hidrogênio , Análise de Sequência de DNA , Virulência , Cultura de Vírus , Replicação Viral
16.
J Gen Virol ; 94(Pt 12): 2691-2699, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24077365

RESUMO

On the basis of generation of an infectious cDNA clone for the BHM26 strain of bovine enterovirus cluster B (BEV-B), 22 sites on different loops of the BHM26 capsid were selected according to an alignment of its sequence with the structural motifs of BEV-A strain VG-5-27 for insertion of the foot-and-mouth disease virus (FMDV) type O-conserved neutralizing epitope 8E8. Two recombinant viruses, rBEV-A1 and rBEV-DE, in which the FMDV epitope was inserted into the VP1 B-C or D-E loops, were rescued by transfection of BHK-21 cells with the in vitro-transcribed RNA of the recombinant BHM26 genome-length cDNA constructs. The two epitope-inserted viruses were genetically stable and exhibited growth properties similar to those of their parental virus in BHK-21 and IBRS-2 cells, which are susceptible to both BEV and FMDV. However, the two recombinant BEVs (rBEVs) had a significantly lower growth titre than those of the parental virus BHM26 in MDBK and Marc145 cells, which are susceptible to BEV but not to FMDV. These results indicated that insertion of the FMDV epitope into the VP1 B-C or D-E loops of the BEV particle altered the replication properties of BEV. In addition, the two rBEVs were sensitive to neutralization by the FMDV type O-specific mAb 8E8, and anti-FMDV IgG antibodies were induced in mice by intramuscular inoculation with the rBEV-A1 and rBEV-DE viruses. Our results demonstrate that the VP1 B-C and D-E loops of the BEV-B particle can effectively display a foreign epitope, making this an attractive approach for the design of BEV-vectored and epitope-based vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Proteínas do Capsídeo/metabolismo , Enterovirus Bovino/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Vetores Genéticos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Bovinos , Linhagem Celular , Enterovirus Bovino/genética , Enterovirus Bovino/metabolismo , Enterovirus Bovino/fisiologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Recombinação Genética , Replicação Viral
17.
J Gen Virol ; 93(Pt 7): 1442-1448, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22513388

RESUMO

Previously, we finely mapped the neutralizing epitopes recognized by foot-and-mouth disease virus (FMDV) type Asia1-specific mAb 3E11 and FMDV type O-specific mAb 8E8. In this study, we engineered recombinant FMDVs of the serotype Asia1 (rFMDVs) displaying the type O-neutralizing epitope recognized by the mAb 8E8. These epitope-inserted viruses were genetically stable and exhibited growth properties that were similar to those of their parental virus. Importantly, the recombinant virus rFMDV-C showed neutralization sensitivity to both FMDV type Asia1 and type O mAbs, as well as to polyclonal antibodies. These results indicated that this epitope-inserted virus has the potential to induce neutralizing antibodies against both FMDV type Asia1 and type O. Our results demonstrated that the G-H loop of FMDV type Asia1 effectively displays the protective neutralizing epitopes of other FMDV serotypes, making this an attractive approach for the design of novel FMDV vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/imunologia , Mutagênese Insercional , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/genética , Epitopos/imunologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/crescimento & desenvolvimento , Testes de Neutralização , Fenótipo , Recombinação Genética
18.
Res Vet Sci ; 124: 178-185, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904721

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Vacinação/veterinária , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Linhagem Celular , Sequência Conservada , Mapeamento de Epitopos , Epitopos/genética , Feminino , Vírus da Febre Aftosa/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas não Estruturais Virais/genética
19.
Front Biosci ; 13: 4707-25, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18508540

RESUMO

Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.


Assuntos
Antivirais/uso terapêutico , DNA Antissenso/uso terapêutico , Genoma Viral , Infecções por Picornaviridae/tratamento farmacológico , Picornaviridae/efeitos dos fármacos , Picornaviridae/genética , RNA Antissenso/uso terapêutico , Antivirais/administração & dosagem , DNA Antissenso/administração & dosagem , Humanos , Lipossomos , Oligonucleotídeos Antissenso/uso terapêutico , Picornaviridae/crescimento & desenvolvimento , Proteoma , RNA Antissenso/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico
20.
Virology ; 518: 87-94, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29455065

RESUMO

Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3Dpol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3Dpol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV.


Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/patogenicidade , RNA Polimerase Dependente de RNA/metabolismo , Substituição de Aminoácidos , Animais , Animais Lactentes , Antivirais/farmacologia , Linhagem Celular , Cricetinae , Fluoruracila/farmacologia , Vírus da Febre Aftosa/efeitos dos fármacos , Regulação Viral da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Plasmídeos , RNA Viral , RNA Polimerase Dependente de RNA/genética , Ribavirina/farmacologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
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