Analysis of cis-acting elements in the 5' leader sequence of alfalfa mosaic virus RNA 3

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem-loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5' terminal 112 nt of this leader contains one of these stem-loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossen et al., Nucleic Acids Res. 21, 1361-1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23-43, 44-90, or 55-112 and inversion or duplication of nucleotides 44-90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60-77) abolished RNA'3 accumulation. Mutations in the stem sections of the putative stem-loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.