Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

Abstract

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (po) is regulated by translational suppression (in-frame readthrough or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded PrilO, and gag-pro-pol-encoded Prl6O polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of PrilO, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Prl1O. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon ofgag or the overlapping CUU codon in thepro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.The genome of the replication-competent retroviruses has most commonly been characterized as a positive-strand RNA of 8.5-9.0 kilobases (kb) that is divided into three genes arranged in the order of 5' gag-pol-env 3' (1). While the protein products of the env gene are translated from a subgenomic (4.0 kb) spliced mRNA, the protein products of gag and gag-pol are synthesized via the translation of the genome-size mRNA. Although the gag-and gag-pol-encoded polyproteins of all known retroviruses are initiated at the same AUG codon of5' gag, the gag-pol precursor is generally expressed at only about 5-20% of the level of the gag precursor. Further, in mammalian type C viruses, the gag and pol gene products are encoded in the same reading frame, but for all other retroviruses, the gag and pol reading frames are out of phase, either overlapping each other or separated by a third reading frame that overlaps both (for review, see ref. 1). Recent investigations led to the structural and functional characterization of viral components encoded between the 3' end of the gag gene and the codon that specifies the N terminus of reverse transcriptase (RT) in the pol gene. Such studies opened ways to elucidating the mechanisms of translational regulation of retroviral protein expression. Thus, reports from this laboratory (2, 3) established that the gag amber terminator of both murine and feline leukemia vir...

A Cytopathological Investigation of Autographa californica Nuclear Polyhedrosis Virus p10 Gene Function Using Insertion/Deletion Mutants

“…Since plaque characteristics were stable and uniform, mutation was not considered a viable explanation. We attribute this expression to a translational frameshift (Hizi et al, 1987;Moore et al, 1987).…”

Stop making sense or Regulation at the level of termination in eukaryotic protein synthesis

“…In principle, frameshifting could involve decoding of a triplet in another reading frame (implying skipping of one or two but not three nt), non-triplet decoding, non-triplet translocation or some other translational quirk [43,71,72]. The data reported above are in favour of another model: a 'slippage model' [52,55]. The frameshifting tRNA would initially bind to its cognate codon in a standard triplet interaction at the ribosomal A site.…”

“…Although the site of frameshifting has been localized in at least two cases [52,55] the exact mechanism of frameshifting is still unclear. In principle, frameshifting could involve decoding of a triplet in another reading frame (implying skipping of one or two but not three nt), non-triplet decoding, non-triplet translocation or some other translational quirk [43,71,72].…”

“…More recently, complete amino acid sequencing of a minor p30 protein found in MMTV virions has shown that this protein is encoded by the gag-X-pro overlap. Alignment of the amino acid sequence of this viral protein with the nucleotide sequence of the MMTV genome, provides a paramount demonstration of the frameshift event in vivo and highlights the site of frameshift [55]. It is interesting to note that in this class of viruses the region where frameshift occurs corresponds to a protein domain (~30 = 'X') different from that of the protease.…”

Frameshifting is required for production of the transposase encoded by insertion sequence 1.

“…To give rise to this transposase in vivo, frameshifting was likely to occur, such that codon 305 (TTA) in the insA frame was recognized as Leu-84 and then codon 307 (AAA) in the B' frame was recognized to give or that codon 308 (AAA) in the insA frame was recognized as Lys-85 and then codon 310 (AAA) in the B' frame was recognized to insert Lys-86. The frameshifting mechanism, by slippage of the tRNA that is reading the 0 frame codon back one nucleotide to the -1 (32,33), human T-cell leukemia virus type II (HTLV-II) (34), and mouse mammary tumor virus (MMTV) (35)(36)(37). The amino acid sequence of the transframe polyprotein encoded by the gag-pro coding region of mouse mammary tumor virus has been determined (37); its translation product is indicated by boldface letters.…”