VIRAL ESCAPE FROM ANTISENSE RNA

RNA coliphage SP was propagated for several generations on a host expr essing an inhibitory antisense RNA complementary to bases 31-270 of th e positive-stranded genome, Phages evolved that escaped inhibition. Ty pically, these escape mutants contained 3-4 base substitutions, but di fferent sequences were observed among different isolates, The mutation s were located within three different types of structural features wit hin the predicted secondary structure of SP genomic RNA: (i) hairpin l oops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule, Computer modelling of the mut ant genomic RNAs showed that all of the substitutions within hairpin s tems improved the Watson-Crick pairing of the stem. No major structura l rearrangements were predicted for any of the mutant genomes, and mos t substitutions in coding regions did not alter the amino acid sequenc e, Although the evolved phage populations were polymorphic for substit utions, many substitutions appeared independently in two selected line s, The creation of a new, perfect, antisense RNA against an escape mut ant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage, Thus, at least in this system, a population of viruses that evolved to escape from a single a ntisense RNA would require a cocktail of several antisense RNAs for in hibition.