RESUMO
We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication.
Assuntos
Antígenos Virais/genética , Vírus da Febre Aftosa/genética , Sinais de Localização Nuclear , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Animais , Antígenos Virais/metabolismo , Linhagem Celular , Núcleo Celular/química , Cricetinae , Análise Mutacional de DNA , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Mutagênese Sítio-Dirigida , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Translation initiation of picornavirus RNA is driven by an internal ribosome entry site (IRES) element located upstream of the initiator codon. RNA structure organization as well as RNA-protein interaction plays a fundamental role in internal initiation. IRES activity has been mainly analyzed in the context of reporter genes, lacking regions of the viral genome potentially affecting translation efficiency. With the aim to understand the vulnerability of the IRES and translation start region to small molecules in the context of the viral genome, we designed a set of customized RNase-resistant 2'O-methyl antisense oligoribonucleotides (2'OMe AONs) based on RNA structure data. These AONs were then used to monitor their capacity to interfere viral RNA translation, and thus, to inhibit virus yield. Foot-and-mouth disease virus (FMDV) RNA translation can be initiated at two in-frame AUG codons. We show here that a 2'OMe AON complementary to AUG2 inhibited viral multiplication more efficiently than the one that targeted AUG1. Furthermore, the response of the viral RNA to AONs targeting the IRES region denoted important differences between tissue culture cells and cell-free systems, reinforcing the need to analyze viral RNA response in living cells. Importantly, we have identified four specific motifs within the IRES element that are targets for viral inhibitors both in tissue culture cells and in cell-free systems. The identified targets define accessible regions to small molecules, which disturb either the RNA structural organization or the RNA-protein interactions needed to initiate translation in FMDV RNA.
Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Ribossomos/genética , Animais , Linhagem Celular , Sistema Livre de Células , Códon , Códon de Iniciação/metabolismo , Cricetinae , Vírus da Febre Aftosa/metabolismo , Genoma Viral , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA Viral/química , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
To clarify the presence of lymphocytic choriomeningitis virus (LCMV) in Spain, we examined blood and tissue specimens from 866 small mammals. LCMV RNA was detected in 3 of 694 wood mice (Apodemus sylvaticus). Phylogenetic analyses suggest that the strains constitute a new evolutionary lineage. LCMV antibodies were detected in 4 of 10 rodent species tested.
Assuntos
Coriomeningite Linfocítica/veterinária , Vírus da Coriomeningite Linfocítica/genética , Murinae , Doenças dos Roedores/virologia , Animais , Coriomeningite Linfocítica/epidemiologia , Coriomeningite Linfocítica/virologia , Filogenia , Estudos Soroepidemiológicos , Espanha/epidemiologiaRESUMO
Recombinant beta-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant beta-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant beta-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.
