Structural Importance of Genomic RNA1 3′ CITE in Blackcurrant Reversion Nepovirus Protein Synthesis

Abstract

Canonical translation initiation and regulation involve end‐to‐end interactions between initiation factors bound to a 7‐methyl guanosine triphosphate cap at the 5′ end and at a poly(A) tail, at the 3′ end of the messenger RNA. This characteristic is also prevalent in many RNA viruses such as the Blackcurrant Reversion Nepovirus (BRV), which has two bipartite genomic RNAs (RNA1 and RNA2) that contain the 3′ poly(A) tail but lack the 5′ cap. The mechanism implemented by BRV during translation initiation is not clearly defined; however, in other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 3′ UTR binds translation initiation factors and delivers the machinery to the 5′ end via proposed RNA‐RNA kissing‐loop interactions. This non‐canonical (cap‐independent) mechanism is directly related to the primary, secondary and higher‐order RNA structure called a cap‐independent translation enhancer (CITE) in the 3′ UTR. Considering the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our goal is to determine the secondary and putative tertiary structure of the 3′ CITE found in the genomic RNA1 in BRV, and correlate the structure to translation initiation function. With this in mind, we identified a set of functionally‐deficient mutants using a luciferase reporter translation assay. We then probed our purified wild‐type and mutant RNAs with a series of covalent nucleobase and sugar backbone modifiers to identify the secondary (and potential tertiary) structure of each RNA. Linking the location of specific modifications to the efficiency of the translation process, our findings show that mutation of a conserved uracil within a highly stable G+C‐rich stem‐loop decreases translation by approximately 40%, but does not significantly alter the structure of the CITE RNA. However, mutating half of the stem to its Watson‐Crick complement significantly alters the RNA1 CITE structure and decreases translation by approximately 80%. From these results, we propose a model whereby the structure and stability of the stem is required to present the loop sequence (including the conserved U) for translation factor recruitment to the viral RNA. Additionally, this data indicates the locations and RNA structures within the CITE directly involved in translation machinery recruitment, allowing for a better understanding and comparison of the tendencies displayed by BRV as well as other positive‐sense RNA plant viruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEM Research Grant MSU Denver College of Letters, Arts and Sciences Dean's Seed Grant MSU Denver Undergraduate Research Conference Student GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.