Probing The Flavivirus Life Cycle: Repurposing Amodiaquine as an Inhibitor of Flavivirus Infectivity and Functional Analysis of Flaviviral NS5 in 5'Capping
Dengue virus serotypes 1-4 (DENV1-4) are transmitted by mosquitoes and are the most frequent cause of arboviral infections in the world. Neither vaccine nor antiviral drug is currently available. In this study, we discovered amodiaquine (AQ), one of 4-aminoquinoline drugs, inhibited DENV2 infectivity with an EC90 of 2.69 ± 0.47 microM and DENV2 RNA replication with an EC50 of 7.41 ± 1.09 microM in the replicon expressing cells. Cytotoxic concentration on BHK-21 cells was 52.09 ± 4.25 microM. The replication inhibition was confirmed by an infectivity assay measured by plaque assay of the extracellular virions, and by qRT-PCR of the intracellular and extracellular viral RNA levels. AQ was stable for at least 96 h and had minor inhibitory effect on entry, translation, and post-replication stages in the viral life cycle. Flaviviral enzymes including protease, methyltransferase, and RNA dependent RNA polymerase do not seem to be targets of AQ. Both p-hydroxyanilino and diethylaminomethyl moieties are important for AQ to inhibit DENV2 replication and infectivity. With a proven efficacy in vivo, AQ will become a new anti-flaviviral candidate for clinical trials.Flaviviral genome contains a 7MeGpppA2'OMe cap structure mimicking that of eukaryotic mRNA so as to evade the host immune system and initiate translation. Synthesis of this cap requires the enzymatic functions of NS3, NS5, and additional unknown factors. Full-length flaviviral NS5 (NS5FL) displayed higher methyltransferase activity than the N-terminal domain alone. Heterologous flaviviral NS5FL proteins were equally active as methyltransferases. A complete understanding of guanylyltransferase activity remains to be explored as NS5 alone is insufficient to add the GMP cap to the 5'-diphosphorylated RNA substrate. An additional cofactor may be required in the transfer of GMP to diphosphorylated RNA, the second step of 5'-capping. Both NS3 and NS5 possess GTP hydrolysis activities resulting in GDP and GMP products, respectively. NS3 is more active than NS5 in GTP hydrolysis such that GDP becomes the predominant product. NS5 acquires higher efficiency in GTP hydrolysis to GMP in the presence of 5'terminal RNAnt1-200. These findings will guide further investigations towards a complete understanding of the flaviviral capping process.
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