Establishment and Application of Flavivirus Replicons.

Abstract

Dengue virus (DENV) and Zika virus (ZIKV) are enveloped, positive-strand RNA viruses belonging to the genus Flavivirus in the family Flaviviridae. The genome of ~11kb length encodes one long open reading frame flanked by a 5' and a 3' untranslated region (UTR). The 5' end is capped and the 3' end lacks a poly(A) tail. The encoded single polyprotein is cleaved co-and posttranslationally by cellular and viral proteases. The first one-third of the genome encodes the structural proteins (C-prM-E), whereas the nonstructural (NS) proteins NS1-NS2A-NS3-NS4A-2K-NS4B-NS5 are encoded by the remaining two-thirds of the genome.Research on flaviviruses was driven forward by the ability to produce recombinant viruses using reverse genetics technology. It is known that the purified RNA of flaviviruses is per se infectious, which allows initiation of a complete viral life cycle by transfecting the genomic RNA into susceptible cells. In 1989, the first infectious flavivirus RNA was transcribed from full-length cDNA templates of yellow fever virus (YFV) facilitating molecular genetic analyses of this virus. In addition to the production of infectious recombinant viruses, reverse genetics can also be used to establish non-infectious replicons. Replicons contain an in-frame deletion in the structural protein genes but still encode all nonstructural proteins and contain the UTRs necessary to mediate efficient replication, a factor that enables their analyses under Biosafety Level (BSL) 1 conditions. This is particularly important since many flaviviruses are BSL3 agents.The review will cover strategies for generating flavivirus replicons, including the establishment of bacteriophage (T7 or SP6) promoter-driven constructs as well as cytomegalovirus (CMV) promoter-driven constructs. Furthermore, different reporter replicons or replicons expressing selectable marker proteins will be outlined using examples of their application to answer basic questions of the flavivirus replication cycle, to select and test antiviral compounds or to produce virus replicon particles. The establishment and application of flavivirus replicons will further be exemplified by my own data using an established YFV reporter replicon to study the role of YFV NS2A in the viral life cycle. In addition, we established a reporter replicon of a novel insect-specific flavivirus, namely Niénokoué virus (NIEV), to define the barrier(s) involved in host range restriction.