PARTIAL CHARACTERIZATION OF A SENDAI VIRUS-REPLICATION PROMOTER AND THE RULE OF 6

We have used a cDNA copy of a natural, internally deleted, Sendai viru s defective interfering genome to study the effect of insertions and d eletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insert ion of 18 nt at nt(72) (in the 5' untranslated region of the N gene, j ust downstream of the le(+) region) was found to be lethal, whereas si milar insertions further from the genome ends were well tolerated. Cur iously, the insertion of 6 nt on either side of the le(+)/N junction ( at nt(47) and nt(67)) was well tolerated, but the insertion of 12 nt a t either site, or of 6 nt at both sites, largely ablated genome amplif ication. These results suggest that an element of this replication pro moter is located downstream of nt(67), in the 5' untranslated region o f the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt(47) plus the insertion of 4, 3, or 2 nt, respecti vely, at nt(67) was poorly tolerated, presumably because the hexamer p hase of the intervening sequence was altered with respect to the N sub units of the template. These results suggest that the rule of six oper ates, at least in part, at the level of the initiation of antigenome s ynthesis. (C) 1996 Academic Press, Inc.