Danny N.P. Doan

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B18NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn2+ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu173 and Pro174 or replacing Pro174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

Structure of the Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus of the Arteriviridae family, genomically related to the coronaviruses. PRRSV is the causative agent of both severe and persistent respiratory disease and reproductive failure in pigs worldwide. The PRRSV virion contains a core made of the 123 amino acid nucleocapsid (N) protein, a product of the ORF7 gene. We have determined the crystal structure of the capsid-forming domain of N. The structure was solved to 2.6 Å resolution by SAD methods using the anomalous signal from sulfur. The N protein exists in the crystal as a tight dimer forming a four-stranded β sheet floor superposed by two long α helices and flanked by two N- and two C-terminal α helices. The structure of N represents a new class of viral capsid-forming domains, distinctly different from those of other known enveloped viruses, but reminiscent of the coat protein of bacteriophage MS2.

Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease

A series of tripeptide aldehyde inhibitors were synthesized and their inhibitory effect against dengue virus type 2 (DENV2) and West Nile virus (WNV) NS3 protease was evaluated side by side with the aim to discover potent flaviviral protease inhibitors and to examine differences in specificity of the two proteases. The synthesized inhibitors feature a varied N-terminal cap group and side chain modifications of a P2-lysine residue. In general a much stronger inhibitory effect of the tripeptide inhibitors was observed toward WNV protease. The inhibitory concentrations against DENV2 protease were in the micromolar range while they were submicromolar against WNV. The data suggest that a P2-arginine shifts the specificity toward DENV2 protease while WNV protease favors a lysine in the P2 position. Peptides with an extended P2-lysine failed to inhibit DENV2 protease suggesting a size-constrained S2 pocket. Our results generally encourage the investigation of di- and tripeptide aldehydes as inhibitors of DENV and WNV protease.

High affinity human antibody fragments to dengue virus non-structural protein 3.

Background The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein. Methodology/Principal Findings In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. Conclusions/Significance Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

Exploring the binding of peptidic West Nile virus NS2B-NS3 protease inhibitors by NMR.

West Nile virus (WNV) NS2B-NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC(50)) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40 kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound 1 (0.23 kcal/mol/HA). The binding of the inhibitors was examined using (1)H-(15)N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the structural domain of the nucleocapsid N protein from porcine reproductive and respiratory syndrome virus (PRRSV)

The structural domain of the PRRSV nucleocapsid N protein was overexpressed in Escherichia coli and purified to homogeneity. Crystals of the expressed protein, designated His-Ndelta(57), were obtained by hanging-drop vapour diffusion using PEG 3350 as precipitant at pH 6.5. A native data set from a frozen crystal was collected to 2.7 A resolution using synchrotron radiation. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = 44.41, c = 125.05, and contain a dimer in the asymmetric unit.

Transplantation of a hydrogen bonding network from West Nile virus protease onto Dengue-2 protease improves catalytic efficiency and sheds light on substrate specificity

The two-component serine protease of flaviviruses such as Dengue virus (DENV) and West Nile virus (WNV) are attractive targets for inhibitor/therapeutic design. Peptide aldehyde inhibitors that bind to the covalently tethered two-component WNV protease (WNVpro) with 50% inhibitory concentration (IC(50)) at sub-micromolar concentrations, bind the equivalent DENV-2 protease (DEN2pro) with IC(50) of micromolar concentrations at best. Conversely, the protease inhibitor aprotinin binds DEN2pro ∼1000-fold more tightly than WNVpro. To investigate the residues that are crucial for binding specificity differences, a binding-site network of hydrogen bonds was transplanted from WNVpro onto DEN2pro. The transplantations were a combination of single, double and triple mutations involving S79D, S83N and S85Q. The mutant DENV proteases, except those involving S85Q, proved to be more efficient enzymes, as measured by their kinetic parameters. The binding affinities of the mutants to peptide inhibitors however showed only marginal improvement. Protein structure modeling suggests that the negatively charged residue cluster, Glu89-Glu92, of the NS2B cofactor may play an important role in determining substrate/inhibitor-binding specificity. These same residues may also explain why aprotinin binds more tightly to DEN2pro than WNVpro. Our results suggest that structure-based inhibitor design experiments need to explicitly consider/include this C-terminal region whose negative charge is conserved across the four DENV serotypes and also among the flavivirus family of proteases.