Partial characterization of a Sendai virus replication promoter and the rule of six

Abstract

We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72 (in the 5' untranslated region of the N gene, just downstream of the le+ region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of the le+/N junction (at nt47 and nt67) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt67, in the 5' untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47 plus the insertion of 4, 3, or 2 nt, respectively, at nt67 was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis.