Recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4A for structural studies.
BACKGROUND
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. 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.


RESULTS
This 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
Small 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.
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Yu-Fu
YF
Hung
Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.
Olga
O
Valdau
Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.
Sven
S
Schünke
Department Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Omer
O
Stern
Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany ; Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Bernd W
BW
Koenig
Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany ; Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Dieter
D
Willbold
Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.
Silke
S
Hoffmann
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D003715:Dengue / Q000517:prevention & control / Q000821:virology; D003716:Dengue Virus / Q000502:physiology*; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D000998:Antiviral Agents; D050505:Mutant Proteins; C080106:NS4A protein, flavivirus; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D017361:Viral Nonstructural Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; C099488:TEV protease