scholarly journals Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum

2002 ◽  
Vol 76 (11) ◽  
pp. 5480-5491 ◽  
Author(s):  
Ivo C. Lorenz ◽  
Steven L. Allison ◽  
Franz X. Heinz ◽  
Ari Helenius
Keyword(s):  
Disulfide Bonds ◽  
Signal Sequence ◽  
Expression System ◽  
Conformational Epitope ◽  
Envelope Proteins ◽  
Viral Glycoprotein ◽  
Virus Envelope ◽  
Folded Structure ◽  
Tick Borne Encephalitis ◽  
Tbe Virus

ABSTRACT Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.

scholarly journals Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion

2004 ◽  
Vol 85 (10) ◽  
pp. 3049-3058 ◽  
Author(s):  
Kentarou Yoshii ◽  
Akihiro Konno ◽  
Akiko Goto ◽  
Junko Nio ◽  
Mayumi Obara ◽  
...  
Keyword(s):  
Point Mutation ◽  
Single Point ◽  
Expression System ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Single Point Mutation ◽  
Virus Envelope ◽  
Electron Microscopic ◽  
Transport Pathway ◽  
Tick Borne Encephalitis

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Acta Naturae ◽  
2015 ◽  
Vol 7 (1) ◽  
pp. 87-97 ◽  
Author(s):  
O. V. Orlova ◽  
V. L. Drutsa ◽  
P. V. Spirin ◽  
A. V. Ivanov ◽  
V. S. Prasolov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Protein Glycosylation ◽  
Virus Envelope ◽  
Mutant Proteins ◽  
Virus Like Particles ◽  
E2 Glycoprotein ◽  
Glycosylation Sites

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

scholarly journals Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

1995 ◽  
Vol 69 (2) ◽  
pp. 695-700 ◽  
Author(s):  
S L Allison ◽  
J Schalich ◽  
K Stiasny ◽  
C W Mandl ◽  
C Kunz ◽  
...  
Keyword(s):  
Envelope Proteins ◽  
Encephalitis Virus ◽  
Acidic Ph ◽  
Virus Envelope ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

scholarly journals Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

2003 ◽  
Vol 77 (7) ◽  
pp. 4370-4382 ◽  
Author(s):  
Ivo C. Lorenz ◽  
Jürgen Kartenbeck ◽  
Anna Mezzacasa ◽  
Steven L. Allison ◽  
Franz X. Heinz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Recombinant Expression ◽  
Side Chain ◽  
Virus Envelope ◽  
Membrane Anchor ◽  
Tick Borne Encephalitis ◽  
Tbe Virus ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

ABSTRACT It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

scholarly journals Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2

2001 ◽  
Vol 82 (7) ◽  
pp. 1629-1635 ◽  
Author(s):  
Laurence Cocquerel ◽  
Jean-Christophe Meunier ◽  
Anne Op de Beeck ◽  
Dorine Bonte ◽  
Czeslaw Wychowski ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Envelope Proteins ◽  
Membrane Insertion ◽  
Virus Envelope ◽  
Glycoprotein Complex ◽  
The Stability ◽  
Major Factors

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel S Yu ◽  
Megan A Outram ◽  
Emma Crean ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

TBE in Croatia

2020 ◽  
Keyword(s):  
Tick Species ◽  
Limited Data ◽  
Notifiable Disease ◽  
Genetic Characteristics ◽  
Central European ◽  
Tick Borne Encephalitis ◽  
Tbe Virus ◽  
Endemic Areas ◽  
European Subtype

Even though tick-borne encephalitis (TBE) has been a notifiable disease in Croatia since 2007, there are no or only limited data available on the occurring tick species in the endemic areas, on the prevalence of TBE virus (TBEV) in ticks, its distribution in Croatia, and its genetic characteristics. Reporting of human cases also is very scarce. The Central European subtype of virus (TBEV-EU) appears to be present in Croatia

TBE in Moldova

2019 ◽  
Author(s):  
Wilhelm Erber ◽  
Tamara Vuković Janković
Keyword(s):  
Reliable Data ◽  
Tick Borne Encephalitis ◽  
Tbe Virus

Although there are no reliable data on the number of tick-borne encephalitis (TBE) cases or the percentage of infected ticks, based on the geography and the presence of TBE virus (TBEV) in all neighboring countries, it must be assumed that TBEV is present anywhere in Moldova.

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