Structure‐Function Analysis of the 3′ CITE from the Blackcurrant Reversion Nepovirus Genomic RNA 2 (BRV2)

Abstract

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Structures within the 3′ UTRs of RNA1 and RNA2 called cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with a region of their 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but this exact mechanism is not well understood. It is thought that the 5′ and 3′ UTRs possess conserved and complementary sequences which facilitate interaction. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to correlate such to its role in translation initiation. Using in silico modeling of folded RNA2 and in vitro RNA structural probing, we determined a preliminary secondary structure for the 3′ CITE. We also identified structural changes when the putative 5′ ‐ 3′ UTR complementarity is disrupted. Using a luciferase reporter gene flanked by the RNA2 3′ CITE and wild‐type BRV2 5′ UTR, we show how the putative 5′ ‐ 3′ UTR complementarity is required for the quantity of and the rate at which luciferase protein is made. We conclude that the 3′ CITE is important for promoting translation initiation and the putative 5′ ‐ 3′ UTR complementarity between the ends of the viral RNA is necessary but not sufficient for protein production. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEMMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.