An N-Terminal Amphipathic Helix in Dengue Virus Nonstructural Protein 4A Mediates Oligomerization and Is Essential for Replication

Abstract

e 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. Dengue virus (DENV) infection is a growing public health threat, with more than one-third of the world population at risk (1). DENV is a positive single-strand RNA virus. Its genome is translated into a single polyprotein, which is cleaved to produce structural (components of the mature virus) and nonstructural (NS) proteins. In addition, the NS proteins generate the viral replication complexes (RC) (2). DENV replicates its RNA genome in association with modified intracellular membranes; the details of the assembly of these complexes are incompletely understood.NS4A, a transmembrane endoplasmic reticulum (ER) resident protein, is thought to induce the host membrane modifications that harbor the viral RC (3). A similar function for NS4A was reported in other flaviviruses (4, 5). To further understand the role of NS4A, we analyzed its cytosolic N-terminal region (amino acids 1 to 48) using sequence alignment of the four DENV serotypes. Within this sequence, amino acids that differed in their identity maintained their biochemical properties, suggesting the presence of a conserved structural motif with a potential functional significance (Fig. 1A). Secondary structure algorithms (6) indicated that this segment is predicted to fold into an ␣-helix (Fig. 1B). Helical wheel projections of amino acids 3 to 20 indicated a conserved polar-nonpolar asymmetry indicative of an amphipathic helix (AH) (Fig. 1C). To experimentally examine the conformation of the NS4A N terminus, a recombinant peptide comprising amino acids 1 to 48 was prepared. Codon-optimized DENV2 NS4A 1-48 was cloned into pGEV2 (7) with an N-terminal fusion to the immunoglobulin binding domain of streptococcal protein G (GB1). A tobacco etch virus (TEV) protease cleavage site (ENLYFQ) was introduced into the beginning of the NS4A coding sequence. Due to difficulties in separating NS4A from GB1 after TEV protease cleavage, an N-terminal glutathione S-transferase (GST) affinity tag was added to GB1-TEV-NS4A(1-48) by cloning it into pGEX4T-2. Protein expression and purification of the GST-GB1-NS4A(1-48) fusion were performed as described previously (8), except that an on-column cleavage was performed by adding TEV protease. The flowthrough was concentrated and subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75) yielding pure NS4A(1-48). The resulting peptide was characterized using far-UV circular dichroism (CD) (Fig. 1D). In aqueous buffer, NS4A(1-48) showed limited solubility and a CD spectrum typical of a random coil conformation with an ␣-helix content below 10%. Addition of various memb...

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

“…6H): helices ␣1 and ␣2 exhibit an amphipathic feature with one surface formed by hydrophobic residues and another surface formed by hydrophilic residues. The above results are in general agreement with a previous report that the first 48 amino acids of DENV-2 NS4A form an amphipathic helix structure (20).…”

“…For NS4A proteins, besides a monomer (17.2 kDa), several SDS-resistant oligomer species (dimer or higher-order oligomers) were detected, suggesting that recombinant NS4A protein can oligomerize in vitro. This result is in agreement with a recent report that the DENV NS4A protein could oligomerize (20). For NS4B protein, two bands, representing a monomer (28 kDa) and dimer (56 kDa), were observed.…”

“…In addition to the DENV-2 NS4A-NS4B interaction, recent studies showed that DENV-2 NS4A or NS4B alone could oligomerize (20,36). The latter findings were confirmed by the current result that recombinant NS4A of DENV-2 formed high-order oligomers and that recombinant NS4B formed dimers on SDS-PAGE gels (Fig.…”

“…The cleavage between NS4A and 2K by NS2B/NS3 proteinase is a prerequisite for the cleavage between 2K and NS4B by a host signalase (18). DENV-2 NS4A consists of 127 amino acids and contains two TMDs (19); the first 48 amino acids of DENV-2 NS4A were reported to form an amphipathic helix that mediates oligomerization (20). DENV-2 NS4B consists of 248 amino acids and contains three TMDs (21).…”

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

“…The DENV NS4A protein has been shown to have an N-terminal amphipathic helix (40), and similar properties are predicted for YFV NS4A. Given the effects of DNAJC14 on regions of the polyprotein in the vicinity of NS4A, we hypothesized that DNAJC14 might bind to an amphipathic helix in NS4A and modulate polyprotein cleavage.…”