Search

There are currently no antiviral therapies available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). In this brief study, we describe the in vitro antiviral activity of adenosine analog NITD008 against KFDV, AHFV, OHFV, as well as Tick-borne Encephalitis virus (TBEV). Alongside the well-established activity of NITD008 against mosquito-borne flaviviruses, our results have demonstrated the feasibility of identifying nucleoside analog inhibitors that have pan-flavivirus activity. [ABSTRACT FROM AUTHOR]

Highlights: [•] We review available structural data on RNA synthesis by flavivirus polymerases. [•] Initiation is the key regulatory step for both flavivirus NS5 and HCV NS5B. [•] How the extra NS5 methyltransferase activity intervenes remains mysterious. [•] NS5 and NS5B activities are prime targets for antiviral drugs. [•] Advanced NS5B inhibitors are put in the context of the enzyme’s structure. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), a mosquito-borne flavivirus, is an important neurotropic human pathogen. As a biosafety level-3 (BSL-3) agent, WNV is strictly to BSL-3 laboratories for experimentations, thus greatly hindering the development of vaccine and antiviral drug. Here, we developed a novel pseudo-infectious WNV reporter virus expressing the Gaussia luciferase (Gluc). A stable 293T NS1 cell line expressing NS1 was selected for trans -supplying NS1 protein to support the replication of WNV-ΔNS1 virus and WNV-ΔNS1-Gluc reporter virus with large-fragment deletion of NS1. WNV-ΔNS1 virus and WNV-Gluc-ΔNS1 reporter virus were confined to complete their replication cycle in this 293T NS1 cell line, displaying nearly identical growth kinetics to WT WNV although the viral titers were lower than those of WT WNV. The reporter gene was stably maintained in virus genome at least within three rounds of passage in 293T NS1 cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo-infectious WNV-Gluc-ΔNS1 could be used for antiviral screening. Furthermore, a high-throughput screening (HTS) assay in a 96-well format was optimized and validated using several known WNV inhibitors, indicating that the optimized HTS assay was suitable for high-throughput screening WNV inhibitors. Our work provides a stable and safe tool to handle WNV outside of a BSL-3 facility and facilitates high throughput screening for anti-WNV drugs. [ABSTRACT FROM AUTHOR]

The flavivirus nonstructural 4B protein (NS4B) has recently emerged as a valid antiviral target for drug discovery. Here we review (i) the current understanding of the structure and function of DENV NS4B, (ii) the approaches that have been taken to identify NS4B inhibitors, and (iii) the known inhibitors of flavivirus NS4B protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. [ABSTRACT FROM AUTHOR]

Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S -adenosyl- L -methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2′- O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket. [ABSTRACT FROM AUTHOR]

Abstract: The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase (RdRp) gene, which is responsible for replication of the viral genome and, as such, is an important target for antiviral therapy. Viral RdRps are known to lack proofreading capabilities and as a result viruses such as WNV exist as a mixture of viral genotypes within an infection, enabling the virus to readily emerge and adapt to new host environments. To test the consequences of subtle structural alterations remote from the RdRp active-site, the following single point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, and T537I; these mutations were selected in an effort to stabilize the secondary structural elements near the rNTP binding pocket of the RdRp. Mutant viruses were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was affected by each mutation and growth and RNA replication kinetics were altered as well. Our results demonstrate that subtle alteration of the RdRp protein away from the active site can have a significant overall biological effect on WNV fitness, and that this effect can be host-dependent. [Copyright &y& Elsevier]

Abstract: Antiviral drug discovery is becoming increasingly important due to the global threat of viral disease pandemics. Many members of the genus Flavivirus are significant human pathogens, among which dengue virus (DENV) alone poses a public health threat to 2.5 billion worldwide, leading to 50–100 million human infections each year. Neither vaccine nor effective therapeutics is currently available for DENV. Development of a DENV vaccine has been challenging, because of the need to simultaneously immunize and induce a long-lasting protection against all four serotypes of DENV; an incompletely immunized individual may be sensitized to life-threatening dengue hemorrhagic fever or dengue shock syndrome. The challenges associated with vaccine development have underscored the importance of development of antiviral therapies for DENV and other flaviviruses. Here we review the strategies to identify inhibitors for DENV therapy. Both viral and host proteins essential for viral replication cycle are potential targets for antiviral development. Inhibitors could be identified by multiple approaches, including enzyme-based screening, viral replication-based screening, structure-based rational design, virtual screening, and fragment-based screening. The strategies discussed in this report should be applicable to antiviral development of other viruses. [Copyright &y& Elsevier]