RESUMO
Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.
Assuntos
Aedes , Dengue , Flavivirus , Replicação Viral , Aedes/virologia , Animais , Dengue/virologia , Flavivirus/metabolismo , Flavivirus/fisiologia , Humanos , Mamíferos , Septinas/genética , Septinas/metabolismoRESUMO
The Flaviviridae family comprises important human pathogens, including Dengue, Zika, West Nile, Yellow Fever and Japanese Encephalitis viruses. The viral genome, a positive-sense single-stranded RNA, is packaged by a single protein, the capsid protein, which is a small and highly basic protein that form intertwined homodimers in solution. Atomic-resolution structures of four flaviviruses capsid proteins were solved either in solution by nuclear magnetic resonance spectroscopy, or after protein crystallization by X-ray diffraction. Analyses of these structures revealed very particular properties, namely (i) the predominance of quaternary contacts maintaining the structure; (ii) a highly electropositive surface throughout the protein; and (iii) a flexible helix (α1). The goal of this review is to discuss the role of these features in protein structure-function relationship.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Flavivirus/metabolismo , Flavivirus/classificação , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Quaternária de Proteína , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The arthropod-borne flaviviruses are important human pathogens, and a deeper understanding of the virus-host cell interaction is required to identify cellular targets that can be used as therapeutic candidates. It is well reported that the flaviviruses hijack several cellular functions, such as exosome-mediated cell communication during infection, which is modulated by the delivery of the exosomal cargo of pro- or antiviral molecules to the receiving host cells. Therefore, to study the role of exosomes during flavivirus infections is essential, not only to understand its relevance in virus-host interaction, but also to identify molecular factors that may contribute to the development of new strategies to block these viral infections. This review explores the implications of exosomes in flavivirus dissemination and transmission from the vector to human host cells, as well as their involvement in the host immune response. The hypothesis about exosomes as a transplacental infection route of ZIKV and the paradox effect or the dual role of exosomes released during flavivirus infection are also discussed here. Although several studies have been performed in order to identify and characterize cellular and viral molecules released in exosomes, it is not clear how all of these components participate in viral pathogenesis. Further studies will determine the balance between protective and harmful exosomes secreted by flavivirus infected cells, the characteristics and components that distinguish them both, and how they could be a factor that determines the infection outcome.
Assuntos
Comunicação Celular , Exossomos/metabolismo , Infecções por Flavivirus/metabolismo , Flavivirus/metabolismo , Interações Hospedeiro-Patógeno , Animais , Vetores Aracnídeos/virologia , Dengue/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Infecções por Flavivirus/transmissão , Humanos , Mosquitos Vetores/virologia , Carrapatos/virologia , Infecção por Zika virus/metabolismoRESUMO
The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.
Assuntos
Infecções por Flavivirus/virologia , Flavivirus/metabolismo , Genes Reporter , Proteínas não Estruturais Virais/metabolismo , Animais , Morte Celular , Linhagem Celular , Vírus da Dengue/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Razão Sinal-Ruído , Zika virus/metabolismoRESUMO
Arboviruses have been a huge threat for human health since the discovery of yellow fever virus in 1901. Arboviruses are arthropod born viruses, mainly transmitted by mosquitoes and ticks, responsible for more than thousands of deaths annually. The Flavivirideae family is probably the most clinically relevant, as it is composed of very important agents, such as dengue, yellow fever, West Nile, Japanese encephalitis, and, recently, Zika virus. Intriguingly, despite their structural and genomic similarities, flaviviruses may cause conditions ranging from mild infections with fever, cutaneous rash, and headache, to very severe cases, such as hemorrhagic fever, encephalitis, Guillain-Barré syndrome, and microcephaly. These differences may greatly rely on viral burden, tissue tropism, and mechanisms of immune evasion that may depend on both viral and host genetic factors. Unfortunately, very little is known about the biology of these factors, and how they orchestrate these differences. In this context, viral structural proteins and host cellular receptors may have a great relevance, as their interaction dictates not only viral tissue tropism, but also a plethora on intracellular mechanisms that may greatly account for either failure or success of infection. A great number of viral receptors have been described so far, although there is still a huge gap in understanding their overall role during infection. Here we discuss some important aspects triggered after the interaction of flaviviruses and host membrane receptors, and how they change the overall outcome of the infection.
Assuntos
Flavivirus/metabolismo , Receptores Virais/metabolismo , Animais , Humanos , Integrinas/metabolismo , Fosfatidilserinas/metabolismoRESUMO
Viruses are important agents of emerging zoonoses and are a substantial public health issue. Among emerging viruses, an important group are arboviruses, which are characterized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of vertebrate hosts. Recently, bats have received increasing attention as an important source for the emergence of zoonoses and as possible viral reservoirs. Among the arboviruses, there are many representatives of the genera Flavivirus and Alphavirus, which are responsible for important epidemics such as Dengue virus, Zika virus and Chikungunya virus. Due to the importance of analyzing potential viral reservoirs for zoonosis control and expanding our knowledge of bat viruses, this study aimed to investigate the presence of viruses of the Alphavirus and Flavivirus genera in bats. We analyzed serum, liver, lungs and intestine from 103 bats sampled in northeast and southern Brazil via Nested-PCR and the hemagglutination inhibition test. All samples tested in this study were negative for arboviruses, suggesting that no active or past infection was present in the captured bats. These data indicate that the bats examined herein probably do not constitute a reservoir for these viruses in the studied areas. Further studies are needed to clarify the role of bats as reservoirs and sources of infection of these viral zoonoses.
Assuntos
Infecções por Arbovirus/patologia , Quirópteros/virologia , Zoonoses/patologia , Alphavirus/genética , Alphavirus/isolamento & purificação , Alphavirus/metabolismo , Animais , Infecções por Arbovirus/virologia , Arbovírus/genética , Arbovírus/isolamento & purificação , Arbovírus/metabolismo , Brasil , Reservatórios de Doenças/virologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Flavivirus/metabolismo , Testes de Inibição da Hemaglutinação , Intestinos/virologia , Fígado/virologia , Pulmão/virologia , RNA Viral/sangue , Zoonoses/virologiaRESUMO
Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE: Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Vírus da Dengue/metabolismo , Dengue/metabolismo , Dengue/virologia , Vitronectina/metabolismo , Linhagem Celular Tumoral , Flavivirus/metabolismo , Humanos , Ligação Proteica/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/metabolismo , Zika virus/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
Flaviviruses include a highly diverse group of arboviruses with a global distribution and a high human disease burden. Most flaviviruses cycle between insects and vertebrate hosts; thus, they are obligated to use different cellular machinery for their replication and mount different mechanisms to evade specific antiviral responses. In addition to coding for viral proteins, the viral genome contains signals in RNA structures that govern the amplification of viral components and participate in triggering or evading antiviral responses. In this review, we focused on new information about host-specific functions of RNA structures present in the 3' untranslated region (3' UTR) of flavivirus genomes. Models and conservation patterns of RNA elements of distinct flavivirus ecological groups are revised. An intriguing feature of the 3' UTR of insect-borne flavivirus genomes is the conservation of complex RNA structure duplications. Here, we discuss new hypotheses of how these RNA elements specialize for replication in vertebrate and invertebrate hosts, and present new ideas associating the significance of RNA structure duplication, small subgenomic flavivirus RNA formation, and host adaptation.
Assuntos
Flavivirus/genética , Interações Hospedeiro-Patógeno/genética , Conformação de Ácido Nucleico , RNA Viral/genética , Adaptação Biológica/genética , Animais , Evolução Molecular , Flavivirus/química , Flavivirus/metabolismo , Genoma Viral , Humanos , Filogenia , RNA Viral/biossíntese , RNA Viral/químicaRESUMO
Hepatitis C, Dengue and West Nile virus are some of the most important flaviviruses, that share one important serine protease enzyme. Serine proteases are the most studied class of proteolytic enzyme and, in these cases, a primary target for drug discovery. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N- t-Boc amino acid esters derived of isomannide as potential serine proteases inhibitors.