Abstract
BackgroundDengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes.Methodology/Principal FindingsThis report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded.Conclusions/SignificanceSmall molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.
Highlights
Dengue fever is currently the fastest-spreading tropical disease in the world, with more than 2.5 billion people at risk
Codon adaption of the non-structural protein 4A (NS4A)(1–48) coding sequence Only negligible amounts of NS4A peptide could be obtained when the original Dengue virus (DENV) type 2 cDNA sequence encoding the first 48 residues of NS4A, NS4A(1–48), was used in a pGEX expression vector. This may be explained by the fact that the viral DNA sequence for NS4A(1–48) is not optimal for expression in E. coli cells, due to different codon usage
The codons were optimized for expression in E. coli cells and chemically synthesized oligonucleotides were used to create a synthetic NS4A(1–48) DNA cassette, which was used to generate several NS4A (1–48) expression constructs
Summary
Dengue fever is currently the fastest-spreading tropical disease in the world, with more than 2.5 billion people at risk. Dengue virus (DENV), the causative agent of this disease, is estimated to infect 390 million people across all continents each year [1]. DENV causes flu-like symptoms in most of the infected patients, but severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. DENV is a positive single strand RNA virus of the Flaviviridae family. Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world’s fastest-spreading tropical disease. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes
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