Mechanistic analysis of the Zika virus translation-replication switch

The genomes of positive-sense RNA viruses are required for both translation and replication during infection. These two processes are antagonistic in nature, each requiring the RNA template in opposite directions. Thus, the balance between these processes during infection must be tightly regulated. Zika virus (ZIKV) is a capped, positive-sense RNA virus of the Flavivirus genus, which contains several notable human pathogens including Dengue virus (DENV) and Japanese encephalitis virus. Infection with ZIKV has been linked to congenital microcephaly as well as Guillain-Barré syndrome in infected adults. During infection, flavivirus replication is known to require genome circularisation, mediated by long-range RNA- RNA interactions between cis-acting elements at the 5′ and 3′ ends of the genome. This facilitates the translocation of the viral polymerase NS5 from its site of recruitment at the 5′ end of the genome to the 3′ end in order to begin negative-strand RNA synthesis. However, it is unknown how the switch from genome translation to genome replication during flavivirus infection is regulated. The work presented for this thesis uses an in vitro reconstitution approach to study translation initiation on ZIKV, in which purified components of the translational machinery are added to the RNA and 48S complex formation upon initiating codons assayed using toeprinting. This technique allows the examination of both recruitment and scanning of the 40S ribosomal subunit, as well as the influence of RNA secondary structure on this process. It is shown that ZIKV translation is cap-dependent using the canonical set of initiation factors and that, under these conditions, recruitment of the viral polymerase to the 5′ proximal stem- loop specifically inhibits translation initiation. Furthermore, circularisation of ZIKV and DENV genomes abrogates viral translation initiation through inhibition of ribosome scanning, subsequently re-directing the ribosome towards upstream near-cognate initiation codons. Conversely, the linear form of the viral genome, which predominates during infection, is shown to be translation-competent. This thesis therefore proposes a model by which the viral polymerase and dynamic viral genome conformations together prime a genome for replication by ensuring ribosome clearance, thus allowing replication to occur unimpeded.Wellcome Trust PhD studentship (105389/Z/14/Z