Minimum requirements for specific binding of RNA and coat protein of alfalfa mosaic virus

Coat protein-protected fragments of alfalfa mosaic virus RNA (AlMV-RNA) and tobacco streak virus RNA (TSV-RNA), which were isolated as described [D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983)Virology, 125, 361-369], were tested for their ability to rebind AlMV coat protein in the presence of an excess of Escherichia coli tRNA by means of a nitrocellulose filter retention assay. In order to obtain the minimum requirements for coat protein binding, a 3'-terminal binding site and several internal binding sites were isolated and fragmented by mild alkali treatment so that various lengths of a particular binding site were present in the mixture to be tested for rebinding capacity. All fragments which originated from the Wend of AlMV-RNA 1 and could bind AlMV coat protein have in common the sequence 5'-CUCAUGCUA-3'. However, this sequence alone is not sufficient to bind viral coat protein. Either an extension by at least 27 nucleotides of this oligomer to the right or an extension by 45 nucleotides (or possibly less) to the left is necessary for AlMV coat protein binding. Also, smaller extensions simultaneously occurring at both sides are sufficient. The smallest fragment which still has binding capacity for viral coat protein is 23 nucleotides long and originates from an internal site of RNA 1. All bound fragments have two common features: the occurrence of AUG(C) twice in the sequence and the potential ability to form a stable secondary structure. A striking observation was that 3'-terminal fragments of TSV-RNAs 1 and 2 rebind AlMV coat protein with low efficiency (about 27 and 37%, respectively), whereas a 3'-terminal fragment of TSV-RNA 3 rebinds AlMV coat protein with an efficiency of about 71%.