Dimerization of flavivirus NS4B protein.

UNLABELLED: Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. IMPORTANCE: The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication.

Conformational flexibility of the Dengue virus RNA-dependent RNA polymerase revealed by a complex with an inhibitor.

We report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery.

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease.

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.