cis-Acting core RNA elements required for negative-strand RNA synthesis and cap-independent translation are separated in the 3′-untranslated region of Red clover necrotic mosaic virus RNA1

The genome of Red clover necrotic mosaic virus (RCNMV) is positive-sense and divided into RNA1 and RNA2. RNA1 has a translation enhancer element (3′ TE-DR1) in the 3′ untranslated region (UTR) that substitutes for a 5′ cap. In this study, we determined the regions required for cap-independent translation and RNA synthesis in the 3′ UTR of RNA1 using a cell-free extract of evacuolated BY-2 protoplasts (BYL) and by an assay in BY-2 protoplasts. The use of capped viral RNA transcripts in the BYL system allowed us to distinguish the effects of introduced mutations on cap-independent translation and negative-strand RNA synthesis of RNA1. We found that the core RNA element of 3′ TE-DR1 essential for cap-independent translation of RNA1 is dispensable for negative-strand RNA synthesis. Thus, cis-acting RNA elements essential for cap-independent translation are separated from those required for negative-strand RNA synthesis in the 3′ UTR of RCNMV RNA1.