牛冠狀病毒天然缺陷株RNA的3’末端基因序列蘊含正股及負股病毒基因體合成所需之Cis-Acting Elements

Abstract

Synthesis of the negative-strand complement of the ~30 kilobase, positive-strand coronaviral genome is a necessary early step for genome replication. It has been shown that for negative-strand RNA synthesis from the mouse hepatitis coronavirus (MHV) genome only the 3’-most 55 nucleotides along with the poly (A) tail in the 3’-untranslated region (UTR) are required. However, map positions for specific cis-acting RNA elements for replication (i.e., both negative and positive-strand RNA synthesis) within this 55-nts region remain unknown. Here, we used a bovine coronavirus defective interfering RNA (DI RNA) with the MHV 3’UTR to characterize the cis-acting RNA requirements in the 3’-terminal 55 nucleotides for viral RNA negative-strand synthesis and replication (interpreted as positive-strand RNA synthesis). The major findings are: (i) Nucleotides from -5 to -14 and -35 to -39 within the 3’-terminal 55 nts are the cis-acting elements responsible for negative-strand BCoV DI RNA synthesis, (ii) Nucleotides from -3 to -34 within the 3’-terminal 55 nts are cis-acting elements required for positive-strand BCoV DI RNA synthesis, (iii) Substitution of 3’-most C residue by U (pyrimidine), but not A and G (purine), is more tolerant for positive-strand synthesis. (iv) Base-paired structure mapping at nt -40 to nt -44 (binding with nt -10 to nt -14) is critical for maintaining efficient positive-strand DI RNA synthesis. The results together demonstrate that the 3’-most 55 nucleotides in the 3’UTR of DI RNA harbor cis-acting elements required not only for negative-strand RNA synthesis but also for positive-strand RNA synthesis.