Structure and biochemical analysis of Kokobera virus helicase

The genus Flavivirus comprises more than 50 RNA virus species that include Yellow fever virus, Dengue virus, Japanese encephalitis virus, and the Tick-borne encephalitis virus complex. Many of these arthropod-borne viruses represent dangerous threats to human health and have been subjected to intensive research to unravel their molecular and virological properties.[1] Kokobera virus was originally found in Northern Queensland in 1960 and is now known to be widespread throughout Australia and Papua New Guinea.[2] Human infections with Kokobera virus occasionally result in an acute polyarticular disease and serological evidence suggests that macropods (kangaroos and wallabies) and horses may be reservoir hosts of this virus.[3] Kokobera virus together with Stratford virus and the recently discovered New Mapoon virus are recognized Australian human pathogenic viruses that are distinct from the Japanese encephalitis virus complex of viruses.[4][5] The flavivirus genomes typically consist of a single positive-sense RNA segment that encodes both structural and replicative proteins. Viral replication takes place in the cytoplasm of the host cell and requires maturation of a polyprotein by viral and endogenous proteases. This process generates a set of nonenzymatic and enzymatic proteins that include an RNA helicase domain, which, like in other flaviviruses, is part of a multifunctional protein named NS3. Because unwinding of the double-stranded RNA molecule is essential for efficient RNA synthesis by the viral polymerase, helicases are considered to be potential targets for antiviral drugs and substantial efforts have been devoted to the development of helicase inhibitors.[6] Phylogenetically, flavivirus helicases belong to a family that is a subset of superfamily 2 helicases.[7] They share seven conserved sequence motifs called I, Ia, Ib, II, III, IV, and V with Motif II featuring the characteristic DEXH box.[8] Here, we report the three-dimensional structure and biochemical investigation of wild-type Kokobera virus helicase and of a mutant targeting a residue of Motif Ia. This study has been carried out within the framework of the Vizier project (www.vizier-europe.org) that is aimed at the molecular characterization of the replicative machinery of RNA viruses. The comparative analysis of the flavivirus helicase structures is the first step towards the development of antiviral compounds interfering with the activity of these essential replicative enzymes.