ALTERATIONS OF THE 3 SHORT OPEN READING FRAMES IN THE ROUS-SARCOMA VIRUS LEADER RNA MODULATE VIRAL REPLICATION AND GENE-EXPRESSION

The Rous sarcoma virus (RSV) leader RNA has three short open reading f rames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiat ion codons in order to prohibit ORF translation, (ii) alterations of t he codon context around the AUG initiation codon to enhance translatio n of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORFI and ORF2 (AUG1 and A UG2) singly or together delayed the onset of viral replication and cel l transformation. In contrast, mutagenesis of AUG3 almost completely s uppressed these viral activities. Mutagenesis of ORF3 to enhance its t ranslation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the in itiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 fro m 7 to 62 codons led to the accumulation of transformation-defective v irus with a delayed onset of replication. In contrast, viruses with el ongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or OR F3 from 9 to 64 codons, without any alterations in the AUG context, ex hibited wild-type phenotypes. These results are consistent with a mode l that translation of the ORFs is necessary to facilitate virus produc tion.