ISOLATION AND CHARACTERIZATION OF VESICULAR STOMATITIS-VIRUS POLR REVERTANTS - POLYMERASE READTHROUGH OF THE LEADER-N GENE JUNCTION IS LINKED TO AN ATP-DEPENDENT FUNCTION

The switch from transcription to replication of the VSV genome is coup led to assembly of nascent chains and involves an unspecified change i n the P-L polymerase complex when it reaches the leader-N gene junctio n. PolR VSV mutants, in contrast to wild-type virus, read through this first gene junction at high frequency without concurrent assembly, an d they show altered ATP requirements for transcription in vitro. The m utation(s) responsible for the polR phenotype segregates to the N-RNA template fraction. We report here that both polR1 and polR2 mutants di splay severe growth restriction in mouse L cells but not in BHK cells. Four of six polR1 revertant viruses, originating from rare plaques on L cells, showed wild-type characteristics for growth, readthrough of leader-N gene junction, and ATP utilization, while two showed partial and quantitatively parallel coreversion of all properties. Sequence an alysis of N and P genes of polR mutants and revertants provided proof that a single mutation in the N protein, Arg179 to His, is responsible for the polR phenotype. PolR1, but not polR2, also displayed a phenot ypically silent GA-to-GG change in the N-P intergenic dinucleotide seq uence. Five of six revertants retained the polR1 N protein mutation an d showed no change in their P gene. We conclude that the L protein lik ely contains second-site suppressors of the polR phenotype, and we pro pose that the switch from transcription to replication is modulated by an ATP-dependent interaction between the template-associated N protei n and the L subunit of the P-L polymerase complex. (C) 1997 Academic P ress.