GENOME NUCLEOTIDE LENGTHS THAT ARE DIVISIBLE BY 6 ARE NOT ESSENTIAL BUT ENHANCE REPLICATION OF DEFECTIVE INTERFERING RNAS OF THE PARAMYXOVIRUS SIMIAN-VIRUS-5

For some members of the Paramyxoviridae family of negative strand RNA viruses, efficient genome replication only occurs when the total genom e length is a multiple of six (6N length, where N is any integer). To determine if this ''rule of six'' requirement applied to the replicati on of the prototype paramyxovirus simian virus 5 (SV5), defective inte rfering (DI) RNA genomes were generated by sequential undiluted passag e of Virus in tissue culture. Molecular cloning and nucleotide sequenc e analysis of 10 RNA genomes revealed a series of copyback DI RNAs wit h chain lengths between 449 and 1365 bases, but only 4 of the 10 natur ally occurring RNA genomes were of 6N length. Many of the cloned DI ge nomes could be grouped into two distinct nested sets, with the members of each set having the same polymerase crossover junctions and extent of terminal complementarity but differing from each other by internal deletions. One of these nested sets of genomes consisted of novel DI RNAs that contained a pentameric stretch of nontemplated adenosine res idues inserted precisely at the polymerase crossover junction. A rever se genetics system was established in which SV5 DI genomes were replic ated in vivo entirely by cDNA-derived components. Using this system, t wo naturally occurring SV5 DI RNAs were examined in a mutational analy sis to determine the role of genome length on SV5 RNA replication. The progressive insertion of one to six nucleotides into a 6N length DI g enome (852 bases) resulted in a reduction in replication for RNAs that contained one to four additional bases (similar to 35-50% of WT level s), followed by an increase back to WT replication levels for genomes that were altered by five and six base insertions (similar to 70 and 1 00% of Wt levels, respectively). An insertion of five nucleotides into a second non-GN length DI RNA (499 total bases) created a genome leng th that was a multiple of six (504 bases) and led to a similar to 10-f old stimulation of replication over that of the unaltered genome. Toge ther, these results indicate that there was a clear influence of 6N ge nome length on SV5 DI RNA replication, but the stringency of this repl ication requirement appeared to be less than that found previously for other paramyxoviruses. This work completes the testing of the rule of six replication requirement for representatives of each of the four g enera of the Paramyxoviridae family and indicates that the preference for replication of 6N length RNA genomes varies between the individual paramyxoviruses. (C) 1997 Academic Press.