Structures and Dynamics of Dengue Virus Nonstructural Membrane Proteins

Abstract

Dengue virus is an important human pathogen threating people, especially in tropical and sub-tropical regions. The viral genome has one open reading frame and encodes one polyprotein which can be processed into structural and nonstructural (NS) proteins. Four of the seven nonstructural proteins, NS2A, NS2B, NS4A and NS4B, are membrane proteins. Unlike NS3 or NS5, these proteins do not harbor any enzymatic activities, but they play important roles in viral replication through interactions with viral or host proteins to regulate important pathways and enzymatic activities. The location of these proteins on the cell membrane and the functional roles in viral replication make them important targets for antiviral development. Indeed, NS4B inhibitors exhibit antiviral activities in different assays. Structural studies of these proteins are hindered due to challenges in crystallization and the dynamic nature of these proteins. In this review, the function and membrane topologies of dengue nonstructural membrane proteins are presented. The roles of solution NMR spectroscopy in elucidating the structure and dynamics of these proteins are introduced. The success in the development of NS4B inhibitors proves that this class of proteins is an attractive target for antiviral development.

Keywords: NMR spectroscopy; antiviral development; dengue virus; drug discovery; membrane protein; membrane topology; nonstructural proteins; protein dynamics.

Figure 3

Structural studies on dengue NS2B. (A) Membrane topology of NS2B on the cell membrane. (B) Model of NS2BFL-NS3 pro. The native form of protease containing full-length NS2B and NS3 protease domain (NS3pro) is shown. NS2B and NS3 are highlighted in green and yellow, respectively. The P1–P4 residues are shown as a sphere structure. (C) The ¹H-¹⁵N-HSQC spectrum of full-length NS2B in detergent micelles. This figure is obtained from [48] with permission. (D) Binding of protease domain to micelles revealed by NMR spectroscopy. Residues such as L31 from NS3 are critical for binding to the membrane. This figure was obtained from [86] with permission.

Figure 1

Organization and membrane topologies of dengue proteins. The viral proteins are highlighted in different color. The NS2B-SN3 protease cleavage sites are indicated as arrows. The functions of NS2A, NS2B, NS4A and NS4B are listed.

Figure 2

Membrane topology of NS2A and residues critical for the function of NS2A. (A) Membrane topology of NS2A. The transmembrane helix with structure determined using NMR is highlighted in brown. (B) Helix view of the three non-transmembrane helices. Helical wheels of these sequence are plotted using DrawCoil 1.0 (https://grigoryanlab.org/drawcoil/ (accessed on 14 February 2022)). (C) NMR structures of pTMD3 in organic system. (D) One structure of pTMS3. The structure (PDB id 2M0S) is shown. The structures were made using PyMOL (https://pymol.org/2/ (accessed on 14 February 2022)). The two positively charged residues in the helix are shown in sticks. More details can be obtained from the reference [57].

Figure 4

Structural studies on dengue NS4A. (A) Membrane topology of NS4A. The helices identified by NMR spectroscopy are shown as cylinders. (B) The ¹H-¹⁵N-HSQC spectrum of full-length NS4A in micelles. (C) Dynamic analysis of NS4A. The dynamics of NS4B shows that these helices are amphipathic or transmembrane helices due to their different relaxation rates. This figure is obtained from the reference [50] with permission.

Figure 5

Structural studies on dengue NS4B. (A) Membrane topology of NS4B based on the NMR studies. The secondary structures of NS4B were determined. (B) ¹H-¹⁵N-HSQC spectra of the N-terminal domain of NS4B and the full-length NS4B in detergent micelles. This figure is obtained from the reference [47,49] with permission.

Figure 6

A simplified flowchart to develop antivirals by targeting these membrane proteins. The key step is to identify hits binding to these proteins, which is challenging as no biochemical assays are available. Deconvolution of hits identified from cell-based assays is needed for understanding the mechanism of action. Using biophysical methods to explore protein–ligand interactions will be useful when recombinant proteins are available.