CIS-ACTING REQUIREMENTS FOR THE REPLICATION OF FLOCK HOUSE VIRUS-RNA 2

To examine the cis-acting requirements for RNA replication, a cDNA clo ne of flock house virus (FHV) RNA 2 was transfected into baby hamster kidney cells and transcribed to yield RNAs that had terminal extension s of different lengths or that lacked internal regions of the molecule . These RNAs were tested for their ability to be replicated by FHV rep licase that was provided by cotransfection of purified FHV RNA 1. The results showed that RNA replication was inhibited by terminal extensio ns, particularly those at the 5' end of the RNA, despite the fact that these extensions were corrected during RNA replication. A negative-se nse transcript with a 12-nucleotide 3' extension was replicated to pro duce a positive-sense RNA that had the correct 5' end, showing that th e replicase could select its correct initiation site from within a lon ger template. A uridine residue at the second position of the positive strand was an important determinant of template activity. RNA molecul es with large internal deletions that amounted to almost 50% of the 1, 400 nucleotides of RNA 2 replicated as efficiently as full-length mole cules, but only if they contained an internal region that lay between nucleotides 538 and 616. Thirty-six spontaneous deletions of RNA 2 tha t arose during sequential replicative passages all conserved the same internal region of the molecule. These results establish that both ter minal and internal regions of FHV RNA 2 play essential roles in making the molecule a competent template for replication.