RESUMO
Mosquito cells allow dengue viruses (DENVs) to undergo replication without causing serious deleterious effects on the cells, leading to advantages for dissemination to other cells. Despite this, increased accumulation of reactive oxygen species (ROS) is usually detected in C6/36 cells with DENV2 infection as shown in mammalian cells. Uniquely, oxidative stress caused by the ROS is alleviated by eliciting antioxidant defense which leads to protection of mosquito cells from the infection. In the present study, a novel p53 paralogue (p53-2) was identified and proved to be regulated in C6/36 cells with DENV2 infection. With a gene-knockdown technique, p53-2 was demonstrated to transcribe catalase which plays a critical role in reducing ROS accumulation and the death rate of infected cells. Ecologically, a higher survival rate of mosquito cells is a prerequisite for prosperous production of viral progeny, allowing infected mosquitoes to remain healthy and active for virus transmission.
Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Proteínas de Insetos/metabolismo , Estresse Oxidativo , Proteína Supressora de Tumor p53/metabolismo , Replicação Viral , Aedes/citologia , Animais , Apoptose , Catalase/genética , Catalase/metabolismo , Contagem de Células , Replicação do DNA , Vírus da Dengue/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The p53 gene is highly important in human cancers, as it serves as a tumor-suppressor gene. Subsequently, two p53 homologues, i.e., p73 and p63, with high identity of amino acids were identified, leading to construction of the p53 family. The p53 gene is highly important in human cancer because it usually transcribes genes that function by causing apoptosis in mammalian cells. In contrast, p63 and p73 tend to be more important in modulating development than inducing cell death, even though they share similar protein structures. Relatively recently, p53 was also identified in mosquitoes and many other insect species. Uniquely, its structure lacks the sterile alpha motif domain which is a putative protein-protein interaction domain and exclusively exists at the C-terminal region in p73 and p63 in mammals. A phylogenetic analysis revealed that the p53 gene derived from mosquitoes is composed of two paralogues, p53-1 and p53-2. Of these, only p53-2 is responsively upregulated by dengue 2 virus (DENV2) in C6/36 cells which usually survive the infection. This indicates that the p53 gene is closely related to DENV infection in mosquito cells. The specific significance of p53-2's involvement in cell survival from virus-induced stress is described and briefly discussed in this report.
Assuntos
Culicidae/genética , Genes p53/genética , Proteínas de Membrana/metabolismo , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Vírus da Dengue/metabolismo , Humanos , Proteínas de Membrana/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Transativadores/genética , Ativação Transcricional/genética , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética , Replicação Viral/genéticaRESUMO
Dengue viruses (DENVs) cause dengue fever which is an important mosquito-borne disease in tropical areas. Generally, DENV does not cause cellular damage in mosquito cells. However, alterations in cytosolic calcium ions ([Ca2+]cyt) and the mitochondrial membrane potential (MMP), as well as accumulated reactive oxygen species (ROS), including superoxide anions (O2â-) and hydrogen peroxide (H2O2), can be detected in C6/36 cells with DENV2 infection. Evident upregulation of BiP/GRP78 also appeared at 24 h postinfection in DENV2-infected C6/36 cells. As expression of BiP/GRP78 mRNA was reduced when the transcription factor X-box-binding protein-1 (XBP1) was knocked down in C6/36 cells, it demonstrated that BiP/GRP78 is the target gene regulated by the XBP1 signal pathway. We further demonstrated that the expression and splicing activity of XBP1 were upregulated in parallel with DENV2 infection in C6/36 cells. In C6/36 cells with BiP/GRP78 overexpression, oxidative stress indicators including [Ca2+]cyt, MMP, O2â-, and H2O2 were all pushed back to normal. Taken together, DENV2 activates XBP1 at earlier stage of infection, followed by upregulating BiP/GRP78 in mosquito cells. This regulatory pathway contributes a cascade in relation to oxidative stress alleviation. The finding provides insights into elucidating how mosquitoes can healthily serve as a vector of arboviruses in nature.
Assuntos
Vírus da Dengue/genética , Dengue/genética , Proteínas de Choque Térmico/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Culicidae/genética , Culicidae/virologia , Dengue/transmissão , Dengue/virologia , Vírus da Dengue/patogenicidade , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/genética , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Superóxidos/metabolismoRESUMO
Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.