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1.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298900

RESUMO

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.


Assuntos
Pestivirus/genética , Proteínas do Envelope Viral/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Dimerização , Mutação/genética , Coelhos , Internalização do Vírus
2.
Viruses ; 13(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201636

RESUMO

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.

3.
J Virol ; 95(15): e0052121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011544

RESUMO

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2. IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.


Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Vírus da Diarreia Viral Bovina/genética , Glicoproteínas de Membrana/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Coelhos , Proteínas do Envelope Viral/genética
4.
Ticks Tick Borne Dis ; 12(4): 101693, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33690089

RESUMO

Tick-borne encephalitis virus (TBEV) is a vector-borne pathogen that can cause serious neurological symptoms in humans. Across large parts of Eurasia TBEV is found in three traditional subtypes: the European, the Siberian and the Far-eastern subtype. Small mammalian animals play an important role in the transmission cycle as they enable the spread of TBEV among the vector tick population. To assess the impact of TBEV infection on its natural hosts, outbred bank voles (Myodes glareolus) were inoculated with one out of four European TBEV strains. Three of these TBEV strains were recently isolated in Germany. The forth one was the TBEV reference strain Neudörfl. Sampling points at 7, 14, 28, and 56 days post inoculation allowed the characterization of the course of infection. At each time point, six animals per strain were euthanized and eleven organ samples (brain, spine, lung, heart, small and large intestine, liver, spleen, kidney, bladder, sexual organ) as well as whole blood and serum samples were collected. The majority of bank voles (92/96) remained clinically unaffected after the inoculation with TBEV, but still developed a systemic infection during the first week, which transitioned to a viraemia and an infestation of the brain in some animals for the remainder of the first month. Viral RNA was found in whole blood samples of several animals (50/96), but only in a small fraction of the corresponding serum samples (4/50). From the whole blood, virus was successfully reisolated in cell culture until 14 days after inoculation. Less than five percent of all inoculated bank voles (4/96) displayed signs of distress in combination with a rapid weight loss and had to be euthanized prematurely. Overall, the recently isolated TBEV strains showed marked differences, such as a more frequent development of long-term viraemia and a higher detection rate of viral RNA in various organs, in comparison to the reference strain Neudörfl. Overall, our data suggest that the bank vole is a potential amplifying host in the TBEV transmission cycle and appears to be highly adapted to circulating TBEV strains.


Assuntos
Arvicolinae , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/veterinária , Doenças dos Roedores/virologia , Animais , Encefalite Transmitida por Carrapatos/virologia , Feminino , Alemanha , Masculino
5.
Pathogens ; 9(10)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987685

RESUMO

West Nile virus (WNV) is a mosquito-borne agent that has also been isolated from several tick species. Vector competence of Ixodes ricinus, one of the most common tick species in Europe, has been poorly investigated for WNV to date. As such, to evaluate the vector competence, laboratory reared Ixodes ricinus nymphs were in vitro fed with WNV lineage 1 infectious blood, allowed to molt, and the resulting females artificially fed to study the virus transmission. Furthermore, we studied the kinetics of WNV replication in ticks after infecting nymphs using an automatic injector. Active replication of WNV was detected in injected nymphs from day 7 post-infection until 28 dpi. In the nymphs infected by artificial feeding, the transstadial transmission of WNV was confirmed molecularly in 46.7% of males, while virus transmission during in vitro feeding of I. ricinus females originating from infected nymphs was not registered. The long persistence of WNV in I. ricinus ticks did not correlate with the transmission of the virus and it is unlikely that I. ricinus represents a competent vector. However, there is a potential reservoir role that this tick species can play, with hosts potentially acquiring the viral agent after ingesting the infected ticks.

6.
Viruses ; 12(5)2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32354202

RESUMO

In 2018, West Nile virus (WNV) broke out for the first time in Germany, with continuation of the epidemic in 2019, involving birds, horses and humans. To identify vectors and characterize the virus, mosquitoes were collected in both years in zoological gardens and on a horse meadow immediately following the diagnosis of disease cases in birds and horses. Mosquitoes were identified and screened for WNV by qRT-PCR, with virus-positive samples being sequenced for the viral envelope protein gene. While no positive mosquitoes were found in 2018, seven mosquito pools tested positive for WNV in 2019 in the Tierpark (Wildlife Park) Berlin. The pools consisted of Cx. pipiens biotype pipiens (n = 5), and a mixture of Cx. p. biotype pipiens and Cx. p. biotype molestus (n = 2), or hybrids of these, and were collected between 13 August and 24 September 2019. The virus strain turned out to be nearly identical to two WNV strains isolated from birds diseased in 2018 in eastern Germany. The findings represent the first demonstration of WNV in mosquitoes in Germany and include the possibility of local overwintering of the virus.


Assuntos
Culicidae/virologia , Doenças dos Cavalos/transmissão , Mosquitos Vetores/virologia , Febre do Nilo Ocidental/transmissão , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/fisiologia , Animais , Doenças das Aves/virologia , Aves/virologia , Culicidae/fisiologia , Alemanha , Doenças dos Cavalos/virologia , Cavalos , Mosquitos Vetores/fisiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética
7.
Viruses ; 12(4)2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32326472

RESUMO

One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 (12 birds, two horses), resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures.


Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Zoonoses Virais/epidemiologia , Zoonoses Virais/virologia , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental , Animais , Teorema de Bayes , Doenças Transmissíveis Emergentes/transmissão , Epidemias , Genoma Viral , Geografia Médica , Alemanha/epidemiologia , Humanos , Filogenia , Filogeografia , Vigilância da População , Medição de Risco , Zoonoses Virais/transmissão , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/genética
8.
Zoonoses Public Health ; 67(4): 416-424, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32162489

RESUMO

West Nile virus (WNV), a zoonotic arbovirus, has recently established an autochthonous transmission cycle in Germany. In dead-end hosts like humans and horses the WNV infection may cause severe symptoms in the central nervous system. In nature, WNV is maintained in an enzootic transmission cycle between birds and ornithophilic mosquitoes. Bridge vector species, such as members of the Culex pipiens complex and Aedes spp., also widely distributed in Germany, might transmit WNV to other vertebrate host species. This study determined and compared the vector competence of field-collected northern-German Cx. pipiens biotype pipiens and laboratory-reared Ae. vexans Green River (GR) for WNV lineage 1 (strain: Magpie/Italy/203204) and WNV lineage 2 (strain: "Austria") under temperatures typical for northern Germany in spring/summer and autumn. For assessment of vector competence, 7- to 14-day-old female mosquitoes were offered a WNV containing blood meal via Hemotek membrane feeding system or cotton-stick feeding. After incubation at 18°C respectively 24°C for 14 days engorged female mosquitoes were salivated and dissected for determination of infection, dissemination and transmission rates by reverse transcriptase quantitative real-time PCR (RT-qPCR). Both Ae. vexans GR and Cx. pipiens biotype pipiens were infected with both tested WNV strains and tested 14 days post-inoculation. Disseminated infections were detected only in Ae. vexans GR incubated at 18°C and in Cx. pipiens pipiens incubated at 24°C after infection with WNV lineage 1. Transmission of WNV lineage 1 was detected in Cx. pipiens pipiens incubated at 24°C. These results indicate that Cx. pipiens pipiens from Northern Germany may be involved in the transmission of WNV, also to dead-end hosts like humans and horses.


Assuntos
Aedes , Culex , Mosquitos Vetores/virologia , Vírus do Nilo Ocidental/classificação , Vírus do Nilo Ocidental/fisiologia , Animais , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Células Vero
9.
Viruses ; 11(11)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31731773

RESUMO

Tick-borne encephalitis is the most important tick-transmitted zoonotic virus infection in Eurasia, causing severe neurological symptoms in humans. The causative agent, the tick-borne encephalitis virus (TBEV), circulates between ticks and a variety of mammalian hosts. To study the interaction between TBEV and one of its suspected reservoir hosts, bank voles of the Western evolutionary lineage were inoculated subcutaneously with either one of eight TBEV strains or the related attenuated Langat virus, and were euthanized after 28 days. In addition, a subset of four strains was characterized in bank voles of the Carpathian linage. Six bank voles were inoculated per strain, and were housed together in groups of three with one uninfected in-contact animal each. Generally, most bank voles did not show any clinical signs over the course of infection. However, one infected bank vole died and three had to be euthanized prematurely, all of which had been inoculated with the identical TBEV strain (Battaune 17-H9, isolated in 2017 in Germany from a bank vole). All inoculated animals seroconverted, while none of the in-contact animals did. Viral RNA was detected via real-time RT-PCR in the whole blood samples of 31 out of 74 inoculated and surviving bank voles. The corresponding serum sample remained PCR-negative in nearly all cases (29/31). In addition, brain and/or spine samples tested positive in 11 cases, mostly correlating with a positive whole blood sample. Our findings suggest a good adaption of TBEV to bank voles, combining in most cases a low virulence phenotype with detectable virus replication and hinting at a reservoir host function of bank voles for TBEV.


Assuntos
Arvicolinae/virologia , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/veterinária , Doenças dos Roedores/virologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Células Cultivadas , Reservatórios de Doenças/virologia , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Humanos , Imunoensaio , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças dos Roedores/diagnóstico , Avaliação de Sintomas
10.
Trop Med Infect Dis ; 4(3)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438608

RESUMO

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016.

11.
J Infect Dis ; 218(suppl_5): S360-S364, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30053054

RESUMO

Work with infectious Ebola virus is restricted to biosafety level (BSL) 4 laboratories. To overcome this limitation, life cycle modeling systems, which recapitulate part or all of the virus life cycle under BSL-1 or -2 conditions, have been developed. The tetracistronic transcription and replication-competent virus-like particle (trVLP) system is currently the most advanced of these systems and is particularly useful for drug screening. However, previous versions have used luciferase reporters, limiting the types of screening assays that can be performed. Here we describe the generation and optimization of a green fluorescent protein-expressing tetracistronic trVLP system, enabling high-content imaging and flow cytometry approaches.Summary: Transcription and replication-competent virus-like particle (trVLP) systems are powerful tools to model the life cycle of highly pathogenic Ebola viruses. Here we describe the generation of a novel, GFP-based trVLP system that allows high content imaging and flow cytometry approaches.


Assuntos
Ebolavirus/genética , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Transcrição Genética/genética , Replicação Viral/genética , Linhagem Celular , Genes Reporter/genética , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos
12.
J Gen Virol ; 98(10): 2482-2494, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28874234

RESUMO

Pestiviruses are enveloped viruses that bud intracellularly. They have three envelope glycoproteins, Erns, E1, and E2. E2 is the receptor binding protein and the main target for neutralizing antibodies. Both Erns and E2 are retained intracellularly. Here, E2 of the bovine viral diarrhea virus (BVDV) strain CP7 was used to study the membrane topology and intracellular localization of the protein. E2 is localized in the ER and there was no difference between E2 expressed alone or in the context of the viral polyprotein. The mature E2 protein was found to possess a single span transmembrane anchor. For the mapping of a retention signal CD72-E2 fusion proteins, as well as E2 alone were analysed. This confirmed the importance of the transmembrane domain and arginine 355 for intracellular retention, but also revealed a modulating effect on retention through the cytoplasmic tail of the E2 protein, especially through glutamine 370. Mutants with a strong impact on retention were tested in the viral context and we were able to rescue BVDV with certain mutations that in E2 alone impaired intracellular retention and lead to export of E2 to the cells surface.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Células CHO , Bovinos , Linhagem Celular , Cricetinae , Cricetulus , Estrutura Secundária de Proteína , Coelhos , Proteínas do Envelope Viral/genética
13.
Sci Rep ; 7(1): 3923, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28634388

RESUMO

Chlamydiae are bacterial pathogens that grow in vacuolar inclusions. Dendritic cells (DCs) disintegrate these compartments, thereby eliminating the microbes, through auto/xenophagy, which also promotes chlamydial antigen presentation via MHC I. Here, we show that TNF-α controls this pathway by driving cytosolic phospholipase (cPLA)2-mediated arachidonic acid (AA) production. AA then impairs mitochondrial function, which disturbs the development and integrity of these energy-dependent parasitic inclusions, while a simultaneous metabolic switch towards aerobic glycolysis promotes DC survival. Tubulin deacetylase/autophagy regulator HDAC6 associates with disintegrated inclusions, thereby further disrupting their subcellular localisation and stability. Bacterial remnants are decorated with defective mitochondria, mito-aggresomal structures, and components of the ubiquitin/autophagy machinery before they are degraded via mito-xenophagy. The mechanism depends on cytoprotective HSP25/27, the E3 ubiquitin ligase Parkin and HDAC6 and promotes chlamydial antigen generation for presentation on MHC I. We propose that this novel mito-xenophagic pathway linking innate and adaptive immunity is critical for effective DC-mediated anti-bacterial resistance.


Assuntos
Ácido Araquidônico/metabolismo , Chlamydia/crescimento & desenvolvimento , Células Dendríticas/citologia , Mitofagia , Fosfolipases A2 Citosólicas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Chlamydia/citologia , Técnicas de Cocultura , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Glicólise , Desacetilase 6 de Histona/metabolismo , Camundongos , Viabilidade Microbiana , Linfócitos T/citologia , Linfócitos T/metabolismo
14.
Methods Mol Biol ; 1499: 15-35, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27987141

RESUMO

Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.


Assuntos
Vírus de RNA/genética , Vírus de RNA/imunologia , RNA Viral/genética , RNA Viral/imunologia , Vacinas/imunologia , Animais , Humanos , Replicon/genética , Replicon/imunologia , Vacinas/genética
15.
J Virol ; 89(20): 10333-46, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26246575

RESUMO

UNLABELLED: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE: HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.


Assuntos
Hepacivirus/química , Proteínas Recombinantes de Fusão/química , Proteínas do Envelope Viral/química , Vírion/química , Motivos de Aminoácidos , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hepacivirus/genética , Hepacivirus/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Vírion/genética , Vírion/ultraestrutura , Montagem de Vírus , Internalização do Vírus
16.
Adv Virus Res ; 93: 47-160, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26111586

RESUMO

Pestiviruses are among the economically most important pathogens of livestock. The biology of these viruses is characterized by unique and interesting features that are both crucial for their success as pathogens and challenging from a scientific point of view. Elucidation of these features at the molecular level has made striking progress during recent years. The analyses revealed that major aspects of pestivirus biology show significant similarity to the biology of human hepatitis C virus (HCV). The detailed molecular analyses conducted for pestiviruses and HCV supported and complemented each other during the last three decades resulting in elucidation of the functions of viral proteins and RNA elements in replication and virus-host interaction. For pestiviruses, the analyses also helped to shed light on the molecular basis of persistent infection, a special strategy these viruses have evolved to be maintained within their host population. The results of these investigations are summarized in this chapter.


Assuntos
Infecções por Pestivirus/veterinária , Pestivirus/genética , Animais , Genoma Viral , Humanos , Gado , Pestivirus/classificação , Pestivirus/isolamento & purificação , Infecções por Pestivirus/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
J Virol ; 86(18): 9606-16, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22740401

RESUMO

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.


Assuntos
Hepacivirus/metabolismo , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Proteínas do Envelope Viral/metabolismo , Sítios de Ligação , Células HEK293 , Hepacivirus/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetraspanina 28/genética
18.
J Biol Chem ; 286(16): 13954-65, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21343309

RESUMO

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.


Assuntos
Antígenos CD/química , Hepacivirus/metabolismo , Hepatite C/metabolismo , Proteínas de Membrana/química , Motivos de Aminoácidos , Animais , Biotinilação , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Citosol/metabolismo , Glicina/química , Humanos , Glicoproteínas de Membrana/química , Ligação Proteica , Estrutura Terciária de Proteína , Tetraspanina 28 , Tetraspanina 29 , Tetraspaninas
19.
Viruses ; 2(8): 1782-803, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21994707

RESUMO

Chronic Hepatitis C infection is a global health problem. While primary infection is often inapparent, it becomes chronic in most cases. Chronic infection with Hepatitis C virus (HCV) frequently leads to liver cirrhosis or liver cancer. Consequently, HCV infection is one of the leading causes for liver transplantation in industrialized countries. Current treatment is not HCV specific and is only effective in about half of the infected patients. This situation underlines the need for new antivirals against HCV. To develop new and more efficient drugs, it is essential to specifically target the different steps of the viral life cycle. Of those steps, the targeting of HCV assembly has the potential to abolish virus production. This review summarizes the advances in our understanding of HCV particle assembly and the identification of new antiviral targets of potential interest in this late step of the HCV life cycle.

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