Flavivirus RNA synthesis in vitro

•Flavivirus RNA replicates in the virus-induced membrane compartments in the cytoplasm.•Methods are given to measure activity of flavivirus RNA polymerase in RNA synthesis in vitro.•We discuss future research perspectives to expand our knowledge in this field.

Establishment of in vitro systems to study mechanisms of RNA synthesis for positive strand RNA viruses have been very useful in the past and have shed light on the composition of protein and RNA components, optimum conditions, the nature of the products formed, cis-acting RNA elements and trans-acting protein factors required for efficient synthesis. In this review, we summarize our current understanding regarding the requirements for flavivirus RNA synthesis in vitro. We describe details of reaction conditions, the specificity of template used by either the multi-component membrane-bound viral replicase complex or by purified, recombinant RNA-dependent RNA polymerase. We also discuss future perspectives to extend the boundaries of our knowledge.