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.METHODOLOGY/PRINCIPAL FINDINGS: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/SIGNIFICANCE: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

Analysis of the Bin1 SH3 interaction with peptides derived from the hepatitis C virus protein NS5A and c-Myc reveals that NS5A can competitively displace c-Myc in vitro

Severe liver damage like cirrhosis and hepatocellular carcinoma (HCC) can be caused by manifestation of the hepatitis C virus (HCV) infection. Constitutively activated c-Myc oncogene has been shown to contribute to the establishment of HCV-mediated HCC. Interestingly, only one of many isoforms of the tumor suppressor protein Bin1 (bridging integrator 1), Bin1+12A, contains an internal, canonical SH3 binding motif that recognizes its own SH3 domain. This leads to the inability of Bin1+12A to interact with c-Myc. The expression of the Bin1+12A isoform is a main phenotype in malignant melanoma cells. We suggest that also other mechanisms that disturb the interaction of Bin1 and c-Myc might have severe consequences since the latter is tightly regulated in healthy cells. The HCV nonstructural protein 5A (NS5A) plays a key role in virus replication and assembly. NS5A plays an intercepting role in several cellular pathways, which are linked to cell growth, cell cycle control, cell survival, cellular stress response, apoptosis as well as HCC. It is known that NS5A contains a highly conserved canonical, polyproline (PxxP) SH3-binding motif, which is located between its D2 and D3 domains. This PxxP motif was described to interact with the SH3 domain of Bin1. In addition to a biophysical analysis of the canonical binding between Bin1 SH3 and the PxxP motif of NS5A [1], we identified two additional low-affinity binding sites for non-canonical SH3 binding on NS5A [2]. The hypothesis underlying the work presented here is that viral NS5A is able to sequester cellular Bin1 from c-Myc

An N-terminal amphipathic helix in the Dengue virus nonstructural protein 4A mediates oligomerization and is essential for replication.

Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy

Primers used for amplification of NS4A (1–48).

<p>Note: The sequences of the restriction sites used for cloning are underlined, the name of the enzyme is given below.</p

Comparative expression of NS4A(1–48) constructs containing single fusion tags.

<p>(A) SDS-PAGE analysis of the relative expression levels of NS4A fusions with ubiquitin (Ubi-NS4A(1–48)), glutathion-S-transferase (GST-NS4A(1–48)) and immunoglobulin-binding domain of streptococcal protein G (GB1-NS4A(1–48)). Aliquots of the expression cultures taken before (0) or 3 hours after IPTG induction (I) were applied. Aliquots of the supernatants after cell lysis (S) are shown as well. (B) TEV cleavage of the purified GB1-NS4A(1–48) fusion protein. Purified GB1-NS4A(1–48) fusion protein after size exclusion chromatography before (−) and after (+) TEV digestion together with a molecular weight marker (M; M3546, Sigma) were applied.</p

Recorded 2D (¹H, ¹⁵N)-BEST-TROSY spectra of the purified NS4A peptides.

<p>Spectrum of 0.5[¹⁵N]-NS4A(1–48) wild type (A) and of 1 mM mutant (L6E, M10E) peptide (B) in 50 mM sodium phosphate buffer, pH 6.8. Data were recorded at 30°C.</p

NS4A(1–48) coding sequence.

<p>ClustalW alignment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086482#pone.0086482-Larkin1" target="_blank">[40]</a> of the NS4A(1–48) coding sequence as found in the viral genome of DENV type 2 and following optimization for <i>E. coli</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086482#pone.0086482-Fath1" target="_blank">[21]</a>.</p

Expression of NS4A(1–48) wild type and mutant peptides using a dual GST-GB1 tag.

<p>(A) Relative expression levels of GST-GB1-NS4A(1–48) and the GST-GB1-NS4A(1–48; L6E, M10E) mutant were analyzed by SDS-PAGE using aliquots of the expression cultures. Shown are samples obtained from culture at 0 (0) or 3 hours (I) following IPTG induction or the supernatant after cell lysis (S). (B) TEV digest of GST-GB1-NS4A(1–48) wild type and mutant protein fusions. Aliquots of GSH-purified supernatants of wild type and mutant fusion proteins before and after TEV cleavage are shown. Note that besides the GST-GB1-NS4A(1–48) full-length product also shorter fragments, likely GST-GB1 and other truncation fragments, marked by asterisks were produced, which are present in the GSH-purified samples already prior to TEV cleavage. Because staining of free NS4A(1–48) peptides is very faint under the conditions used, the progress of the TEV digest is monitored by observing the decrease of the band for the dual tagged GST-GB1-NS4A(1-48) fusion protein in parallel with an increase of the band for the free GST-GB1 dual tag. A densitometric analysis of the respective bands revealed a cleavage efficiency of approximately 50% for the wild type peptide.</p