Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.
BVDV, bovine viral diarrhea virus; C, capsid protein; CSFV, classical swine fever virus; CCHFV, Crimean-Congo hemorrhagic fever virus; CPE, cyto-pathogenic effect; dsRNA, double-stranded RNA; ER, endoplasmic reticulum; E, envelope protein; GMP, guanosine monophosphate; GTP, guanosine triphosphate; GTase, guanylyltransferase; NS3Hel, helicase; HIV, Human Immunodeficiency Virus I; HCV, hepatitis C virus; HBS, high affinity binding site; IMP, Inosine 5′-monophosphate; LBS, low-affinity binding site; M, membrane protein; NS5MTase, methyltransferase; N7MTase, (guanine-N7)-methyltransferase; 2′OMTase, (nucleoside-2′-O-)-methyltransferase; NS, non-structural; NLS, nuclear localization sequences; NS3Pro, protease; RC, replication-competent complex; RSV, respiratory syncytial virus; NS5RdRp, RNA-dependent RNA polymerase; NS3RTPase, RNA triphosphatase; AdoMet, S-adenosyl-L-methionine; ssRNA, single-stranded RNA; T-705 RMP, T-705-ribofuranosyl-5′-monophosphate; VIZIER, Viral Enzymes Involved in Replication; Flavivirus; Flaviviral NS3 protein; Flaviviral NS5 protein; Protease; Helicase; Polymerase; Methyltransferase; Flavivirus protein structure; Antivirals; VIZIER Consortium
The C-terminal 440 amino acids of the NS3 protein from Kunjin virus (Flaviviridae) code for a helicase. The protein has been overexpressed and crystallized. Characterization of the isolated monoclinic crystal form and diffraction data (at 3.0 Å resolution) are presented, together with a preliminary molecular-replacement solution.
Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3′ nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds.
Flaviviridae; Kunjin virus; West Nile virus; helicases; viral enzymes
Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes.
Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.
(nucleoside-2′-O-)-methyltransferases; flaviviruses; Meaban virus; Yokose virus