Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins

Abstract

The primary translation products of retroviral pol genes are polyproteins initiated in an upstream gene (gag). To investigate the manner in which the gag-initiated polyproteins of the mouse mammary tumor virus are produced, we determined the nucleotide sequence of a 1.8-kilobase DNA fragment that spans the region between gag and pol in the C3H strain of mouse mammary tumor virus. The sequence reveals three overlapping open reading frames: the first encodes products of gag (p27gag and p14gag); the second encodes a protein domain of unknown function (termed X) that is highly related to a similarly positioned sequence in simian type D retroviruses and the viral protease (pro); and the third encodes the reverse transcriptase. The reading frames are organized to permit uninterrupted readthrough from gag to pol if ribosomal frameshifts occur in the -1 direction within each of the two overlapping regions, one of which is 16 nucleotides in length and the other 13 nucleotides. Cell-free translation of RNA containing these overlap regions shows that fusion of the reading frames by ribosomal frameshifting occurs efficiently: about one-fourth of the ribosomes traversing the gag-X/pro overlap and one-tenth traversing the X/pro-pol overlap shift frames, generating gag-related polyproteins in ratios similar to those observed in vivo. Synthetic oligonucleotides containing either of the overlap regions inserted into novel contexts do not induce frameshifting; hence the overlapping portions of the reading frames are not sufficient to induce a frameshift event, and a larger sequence context or secondary structure may be implicated.