cis-acting requirements for the replication of flock house virus RNA 2

To examine the cis-acting requirements for RNA replication, a cDNA clone of flock house virus (FHV) RNA 2 was transfected into baby hamster kidney cells and transcribed to yield RNAs that had terminal extensions of different lengths or that lacked internal regions of the molecule. These RNAs were tested for their ability to be replicated by FHV replicase that was provided by cotransfection of purified FHV RNA 1. The results showed that RNA replication was inhibited by terminal extensions, particularly those at the 5' end of the RNA, despite the fact that these extensions were corrected during RNA replication. A negative-sense transcript with a 12-nucleotide 3' extension was replicated to produce a positive-sense RNA that had the correct 5' end, showing that the replicase could select its correct initiation site from within a longer template. A uridine residue at the second position of the positive strand was an important determinant of template activity. RNA molecules with large internal deletions that amounted to almost 50% of the 1,400 nucleotides of RNA 2 replicated as efficiently as full-length molecules, but only if they contained an internal region that lay between nucleotides 538 and 616. Thirty-six spontaneous deletions of RNA 2 that arose during sequential replicative passages all conserved the same internal region of the molecule. These results establish that both terminal and internal regions of FHV RNA 2 play essential roles in making the molecule a competent template for replication.