Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

Hung, Yu-Fu; Valdau, Olga; Schünke, Sven; Stern, Omer; Koenig, Bernd W.; Willbold, Dieter; Hoffmann, Silke

doi:10.1371/journal.pone.0086482

Cited by 7 publications

(7 citation statements)

References 40 publications

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“…In agreement with these predictions, the N-terminal cytoplasmic tail of the NS4A protein (residues 1-48) was shown to be mostly disordered in solution, but was able to form an amphipathic a-helix (i.e. a-helical protein regions in which one face of the helix is hydrophobic while the opposite face is hydrophilic) in the presence of membranes [138,139]. The DENV NS4B protein was recently shown to homodimerize in vitro, as evidenced by gel filtration, chemical cross-linking, and multi-angle light scattering experiments [140].…”

Section: Non-structural Proteins Ns4a and Ns4bmentioning

confidence: 56%

Unstructural biology of the dengue virus proteins

et al. 2015

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In this study, we used a wide spectrum of bioinformatics techniques to evaluate the extent of intrinsic disorder in the complete proteomes of genotypes of four human dengue virus (DENV), to analyze the peculiarities of disorder distribution within individual DENV proteins, and to establish potential roles for the structural disorder with respect to their functions. We show that several proteins (ER, E, 1, 2A and 4A) are predicted to be mostly ordered, whereas four proteins (C, 2k, NS3 and NS5) are expected to have high disorder levels. The profiles of disorder propensities are similar across the four genotypes, except for the NS5 protein. Cleavage sites are depleted in polymorphic sites, and have a high propensity for disorder, especially relative to neighboring residues. Disordered regions are highly polymorphic in type 1 DENV but have a relatively low number of polymorphic sites in the type 4 virus. There is a high density of polymorphisms in proteins 2A and 4A, which are depleted in disorder. Thus, a high density of polymorphism is not unique to disordered regions. Analysis of disorder/ function association showed that the predominant function of the disordered regions in the DENV proteins is protein-protein interaction and binding of nucleic acids, metals and other small molecules. These regions are also associated with phosphorylation, which may regulate their function.Abbreviations CLV, cleavage site; DENV, dengue virus; ELM, eukaryotic linear motif; IDP, intrinsically disordered protein; IDPR, intrinsically disordered protein region; MoRF, molecular recognition feature; NS, non-structural protein. IntroductionDengue fever virus (DENV) is a member of the family Flaviviridae in the genus Flavivirus, that, among other members, includes hepatitis C virus, West Nile virus and yellow fever virus [1]. The Flavivirus genus consists of nearly 80 viruses, many of which are arthropodborne human pathogens that cause a variety of diseases, including dengue fever, plus the associated dengue hemorrhagic fever and dengue shock syndrome, Japanese encephalitis and yellow fever [2]. There are four antigenically related serotypes of the dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4). All four serotypes are known to cause the full spectrum of disease [3]. Infection with one of these serotypes provides immunity for life, but to only that serotype [4]. Therefore, persons living in a dengue endemic area are at risk of encountering secondary infection with other DENV serotypes.DENV is an arbovirus (arthropod-borne virus) that is primarily transmitted between humans and Aedes aegyptim which breed in domestic and peridomestic water containers. A sylvatic cycle (whereby jungle primates and mosquito vectors perpetuate the virus) has been documented in Southeast Asia and West Africa, but it is presently uncertain to what extent this cycle contributes to human infections [5]. It is believed that this virus displays enzootic maintenance cycles that involve Aedes mosquitoes, which breed in tree holes and transmit the virus between mon...

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Section: Non-structural Proteins Ns4a and Ns4bmentioning

confidence: 56%

Unstructural biology of the dengue virus proteins

et al. 2015

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“…One of them is the B1 domain of streptococcal protein G (56 aa), which forms a compact fold with high solubility, which can contribute to enhanced solubility to the GB1 fused target protein in E . coli ( Bauer et al, 2009 ; Hung et al, 2014 ). GB1 contains two separable domains: N-terminal (1–40 aa) and C-terminal (41–56 aa) domains.…”

Section: Discussionmentioning

confidence: 99%

“…The stability of recombinant proteins in a foreign environment can cause limitations in the increase in production levels. The fusion of soluble tags to recombinant proteins is a promising strategy that has been used in the production of bioactive proteins ( Esposito and Chatterjee, 2006 ; Hung et al, 2014 ). The fusion of foreign domains can lead to an increase in production yield.…”

Section: Introductionmentioning

confidence: 99%

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants

et al. 2022

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Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a “factory” to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.

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“…A mutant peptide containing the mutations L6E and M10E was designated NS4A(1–48, L6E;M10E). The two NS4A peptides were recombinantly produced in E. coli BL21 cells and enzymatically cleaved from the affinity tag as described earlier [ 13 ]. Uniform isotope labeling with 15 N or 13 C, 15 N was achieved by expression in M9 medium containing 15 N ammonium chloride and 13 C glucose (Eurisotop, Saarbrücken, Germany) as the sole source of nitrogen and carbon, respectively.…”

Section: Methodsmentioning

confidence: 99%

Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

Hung

Schwarten

Hoffmann

et al. 2015

Viruses

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Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1–48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.

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Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

Cited by 7 publications

References 40 publications

Unstructural biology of the dengue virus proteins

Unstructural biology of the dengue virus proteins

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants

Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

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