ABSTRACT
The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other.
Subject(s)
Agmatine/chemistry , Dipeptides/chemistry , Encephalitis Virus, Murray Valley/enzymology , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemistry , Viral Nonstructural Proteins , West Nile virus/enzymology , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistryABSTRACT
Murray Valley encephalitis is an infectious disease spread by a mosquito-borne virus endemic in Papua New Guinea and northern Australia. In the past decade, it has spread to various regions of Australia and there is currently no therapeutic treatment against this disease. An attractive drug target is the viral serine protease NS2B/NS3, a critical enzyme involved in viral replication. Herein, we report the inhibitory activities of 37 C-terminal agmatine peptidomimetic inhibitors which led to the design of a novel structurally-constrained competitive inhibitor 38 possessing a Ki of 2.5±0.5 µM. We believe our data provides crucial insights into the viral protease active site specificity which could be used to facilitate drug design against Murray Valley encephalitis viral infections.
Subject(s)
Encephalitis Virus, Murray Valley/enzymology , Peptidomimetics/pharmacology , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation/drug effects , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Peptidomimetics/chemical synthesis , Peptidomimetics/chemistry , Serine Proteinase Inhibitors/chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
BACKGROUND: Usutu virus belongs to the Flaviviridae viral family and constitutes an important pathogen. The viral helicase is an ideal target for inhibitor design, since this enzyme is essential for the survival, proliferation and transmission of the virus. RESULTS: Towards a drug-design approach, the 3D model of the Usutu virus helicase structure has been designed, using conventional homology modelling techniques and the known 3D-structure of the Murray Valley Encephalitis virus helicase, of the same viral family, as template. The model was then subjected to extended molecular dynamics simulations in a periodic box, filled with explicit water molecules for 10 nanoseconds. The reliability of the model was confirmed by obtaining acceptable scores from a variety of in silico scoring tools, including Procheck and Verify3D. CONCLUSION: [corrected] The 3D model of the Usutu virus helicase exhibits in silico all known structural characteristics of the Flaviviridae viral family helicase enzymes and could provide the platform for further de novo structure-based design of novel anti-Usutu agents.
Subject(s)
Flaviviridae/enzymology , Models, Molecular , RNA Helicases/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Computer Simulation , Encephalitis Virus, Murray Valley/enzymology , Encephalitis Virus, Murray Valley/genetics , Flaviviridae/genetics , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Helicases/genetics , RNA Helicases/metabolism , Sequence Homology, Amino Acid , Static Electricity , Structural Homology, Protein , ThermodynamicsABSTRACT
Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation.
Subject(s)
Encephalitis Virus, Murray Valley/enzymology , Models, Molecular , RNA Helicases/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Crystallography, X-Ray , Molecular Sequence Data , Nucleotides/chemistry , RNA Helicases/metabolism , Sequence Alignment , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
We have determined the high resolution crystal structure of the methyltransferase domain of the NS5 polypeptide from the Murray Valley encephalitis virus. This domain is unusual in having both the N7 and 2'-O methyltransferase activity required for Cap 1 synthesis. We have also determined structures for complexes of this domain with nucleotides and cap analogues providing information on cap binding, based on which we suggest a model of how the sequential methylation of the N7 and 2'-O groups of the cap may be coordinated.
