RESUMO
Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Cromatografia em Gel , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Zika virus/genéticaRESUMO
Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.
Assuntos
Vírus da Dengue/genética , Vírus da Dengue/imunologia , Dengue/imunologia , Peptídeos/imunologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Anticorpos Antivirais/metabolismo , Sequência de Bases , Sequência Conservada , Cristalografia por Raios X , Dengue/virologia , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Sorogrupo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
The present study reports the first outbreak of autochthonous canine visceral leishmaniasis in Florianópolis, Santa Catarina, southern Brazil. Following the report of two cases of CVL, the Control Center of Zoonotic Diseases conducted a serological survey by ELISA and IFAT assays in seven districts of the Santa Catarina Island. Eleven seropositive dogs of autochthonous transmission were used in the present study. Infection by Leishmania sp. was confirmed by parasitological examination of bone marrow, liver, spleen and lymph nodes, culture in Schneider's medium and PCR. Leishmania sp. isolates were characterized by PCR-RFLP and hybridization with specific probes, allowing for the identification of Leishmania infantum. Autochthonous transmission of this disease in an area with high tourist traffic presents a major public health concern and signifies the emergence of an important zoonosis in southern Brazil. Therefore, the implementation of surveillance and control measures is imperative to prevent the spread of the disease among the canine population as well as transmission to the human population.
O presente estudo relata o primeiro surto autóctone de leishmaniose visceral canina (LCV) em Florianópolis, Santa Catarina, Brasil. Durante levantamento soro-epidemiológico realizado pelo Centro de Controle de Doenças Zoonóticas (CCZ) envolvendo 2.124 cães, 29 (1,37%) foram soropositivos para VL (ELISA + RIFI). Onze cães positivos por transmissão autóctone foram utilizados no presente estudo. A confirmação da infecção por Leishmania sp. foi realizada pelo exame parasitológico da medula óssea, fígado, baço e linfonodos, cultura em meio Schneider e PCR. Os isolados de Leishmania sp. foram caracterizados por PCR-RFLP e hibridação com sondas específicas, permitindo a identificação de Leishmania infantum. A transmissão autóctone da LCV em uma área com grande fluxo turístico como Florianópolis representa um preocupante risco à saúde pública e o surgimento de uma importante zoonose no sul do Brasil. Neste contexto, a implementação de medidas de vigilância e controle da doença são fundamentais para evitar a propagação da doença entre a população canina, bem como a transmissão para a população humana.