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1 Ribosomal Frameshift in a Double-Stranded RNA Virus of Yeast Forms a Gag-Pol Fusion Protein (RNA Polymerase/Lacz Fusion/L-A Virus) JONATHAN D

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1 Ribosomal Frameshift in a Double-Stranded RNA Virus of Yeast Forms a Gag-Pol Fusion Protein (RNA Polymerase/Lacz Fusion/L-A Virus) JONATHAN D Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 174-178, January 1991 Biochemistry A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein (RNA polymerase/lacZ fusion/L-A virus) JONATHAN D. DINMAN, TATEO ICHO*, AND REED B. WICKNER Section on the Genetics of Simple Eukaryotes, Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Herbert Tabor, September 20, 1990 (received for review August 3, 1990) ABSTRACT The L-A double-stranded RNA (dsRNA) vi- 9, 23). RNA secondary structure downstream of the slippery rus of Saccharomyces cerevisiae has two open reading frames site may slow or stall ribosomes such that they remain in the (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). slippery site longer, thus promoting frameshifting (4). ORF2, which is expressed only as a 180-kDa fusion protein with The L-A genome (Fig. 1A) has two open reading frames ORF1, encodes a single-stranded RNA-binding domain and has (ORFs). ORF1 encodes the 80-kDa major coat protein (anal- the consensus sequence for RNA-dependent RNA polymerases ogous to retroviral gag). ORF2 has a sequence pattern typical of(+)-strand and double-stranded RNA viruses (pol). We show of the RNA-dependent RNA polymerases of (+) ssRNA that the 180-kDa protein is formed by -1 ribosomal frame- viruses and double-stranded (ds) RNA viruses (analogous to shifting by a mechanism indistinguishable from that of retro- pol) and a ssRNA binding activity thought to be involved in viruses. Analysis of the "slippery site" suggests that a low the packaging process (26-30). Fusion of ORF1 and ORF2 probability of unpairing of the aminoacyl-tRNA from the produces the 180-kDa gag-pol-like protein. ORF1 and ORF2 0-frame codon at the ribosomal A site reduces the efficiency of overlap by 130 base pairs (bp) and ORF2 is in the -1 frame frameshifting more than the reluctance ofa given tRNA to have with respect to ORF1. It has been proposed that a -1 its wobble base mispaired. Frameshifting of L-A requires a ribosomal frameshifting event at the site in the overlap region pseudoknot structure just downstream of the shift site. The diagrammed in Fig. 1B results in production of the 180-kDa efficiency of the L-A frameshift site is 1.8%, similar to the viral protein (26). observed molar ratio in viral particles of the 180-kDa fusion We present strong evidence that -1 ribosomal frameshift- protein to the major coat protein. ing fuses ORF1 and ORF2 and analyze the RNA sequences responsible. Frameshifting of L-A requires the predicted The pol genes of retroviruses are expressed as gag-pol or heptamer and a pseudoknot structure that involves the pre- gag-pro-pol fusion polyproteins (1) formed either by in-frame dicted stem-loop structure. We suggest that weak mRNA- read-through of termination codons (2, 3) or by ribosomal tRNA interactions at the ribosomal A site are required for frameshifting (4-6). Both mechanisms allow for production frameshifting. of multiple proteins from a single, unmodified mRNA. In Rous sarcoma virus (RSV), gag and pol genes overlap, with pol being in the -1 frame with respect to gag (7). In 5% MATERIALS AND METHODS oftranslations, a -1 frameshifting event allows ribosomes to Strains. Yeast strain 2907 (MATa his3-d200 1eu2- trpl-d901 miss the gag termination codon and continue to translate the ura3-52 ade2-10 K-) was used for transformation (31). pol gene, producing a gag-pol fusion protein (8, 9). A -1 Strains were grown on YPAD broth or complete synthetic trp ribosomal frameshifting has also been described in corona- medium (H-trp) (32). viruses [(+) single-stranded (ss) RNA genomes] (10, 11), Enzymes and Plasmid Constructions. Plasmid construction phage T7 (12), and in the dnaX gene of Escherichia coli (33), use ofthe Muta-Gene in vitro mutagenesis kit (Bio-Rad) (13-15). A +1 ribosomal frameshift is seen in the yeast (34), and sequencing of dsDNA plasmids with modified T7 retrotransposon Tyl (16-18) and in the E. coli release factor DNA polymerase (35) (Sequenase V.2.0; United States Bio- 2 (19-21). chemical) were by standard procedures. The signals responsible for -1 ribosomal frameshifting The parent expression plasmid, p375, is derived from include a "slippery site" heptamer, X XXY YYZ (gag reading YEpIPT (36) and was obtained from Genentech. p375 contains frame indicated; X = A, U, or G; Y = A or U; Z = A, U, or the following: an ori and the P3-lactamase gene from pBR322, C), followed by a stem-loop structure that can be involved in the yeast TRPI gene, the ori of the 2-,u yeast plasmid, and a an RNA pseudoknot (4, 9, 13, 20, 22, 23). A pseudoknot is polylinker 3' of the yeast PGKI promoter. p375 was modified base pairing ofthe loop with a sequence 3' ofa stem-loop (24, to include a translational start site 3' of the PGKI promoter 25). The "simultaneous slippage" model of Jacks et al. (4) and 5' of the polylinker to create pTI21. pTI21 was digested proposes that the tRNAs bound at the ribosomal P site to with Sst I and S1 nuclease and the BamHI lacZ fragment from XXY and at the A site to YYZ simultaneously slip back 1 base pMC1790 (37) treated with T4 DNA polymerase was inserted on the mRNA to pair with XXX and YYY, respectively. to produce pTI23. pTIL121 contains the EcoRI/Pst I fragment Because their nonwobble bases remain properly paired, this of L-A (bases 1763-2122) including the region of overlap of can happen at a finite rate (Fig. 1). The stem-loop structure ORF1 and ORF2 inserted into the Bluescript SK+ vector has been demonstrated to be essential for efficient frame- (Stratagene) cut with the same enzymes. pTI23 was cleaved at shifting in RSV (4), infectious bronchitis virus (23), and the the BamHI and Kpn I sites in the polylinker and three sets of E. coli dnaX gene (13) and is predicted to occur following the oligonucleotide linkers were inserted to generate pTI24 (lacZ slippery site heptamers of a number of other retroviruses (4, Abbreviations: RSV, Rous sarcoma virus; ss, single stranded; ds, The publication costs of this article were defrayed in part by page charge double stranded; ORF, open reading frame; ,3-gal, B3-galactosidase. payment. This article must therefore be hereby marked "advertisement" *Present address: Tokyo Medical and Dental University, Tokyo, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Japan. 174 Downloaded by guest on September 30, 2021 Biochemistry: Dinman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 175 A Two L-A ORFs Encode Chimeric RNA Polymerase - of 5, 11, and 17 codons of the 5' portion of the L-A-derived RNA Binding Protein with Major Coat Protein Domain cDNA sequence (Figs. 2 and 3). The pF'8-3D series lacked 32 and 38 codons of L-A cDNA sequence from the 3' end and 2072 Encapsidation included a termination codon in the 0 frame downstream of 1939 Signal 4546 the shift site. pF'8-5D17/3D32XS was constructed from 30 IRE ^\ _ 4579 pF'8-5D17 to produce a 32-codon deletion of L-A cDNA 5, _ | ~~~3, sequence information from the 3' region. Changes made in site for the slippery site of pF'8 are shown in Fig. 4. In the text, the ORF1 = gag replication triplets are shown with the ORF1 frame indicated by a space. ORF2 = poi pJD18 had the complement of the 5' pseudoknot region -1 ribosomal framreshifting site (GCCAGC -* CGGUCG), and pJD19 had the complement of 80 kDa the 3' pseudoknot-forming region (GCUGGC -> CGACCG) 2::;: ........::.. 2: ........... .:......... major coat protein RNA RNA (Figs. 2 and 3). pJDRPsi used pJD19 and the mutagenic 180 kDa binding polymerase oligonucleotide used to create pJD18 to construct the double coat protein mutant. domain In pJD20, the U residue 3' of the loop (base 1991) was a C. In pJD21, the U residue in the bulge of the stem (base 1996 B %IN of L-A) was a G. pJD17 incorporated both changes (see Figs. Pseudoknot IC and 2). fi-Galactoside (fl-gal) Activity. Assays of permeabilized yeast cells were as described (39). Cells were grown in H-trp ...GGGUUUAGG...IL medium to midlogarithmic phase, and assays were normal- ORF1 (gag) ,II L I I...-..pJ ized to the OD6w of the culture and to the assay time. Three individual isolates of each mutant were assayed in triplicate. Control experiments with pF8 and pTI25 showed that their C L-A relative and absolute ,8-gal activity did not vary with phase of ORF1-ORF2 PGK the growth cycle (data not shown). overlap 2p p LacZ Origin TRP1 peR322 _ reglon- m 0 m 0 m m m AUG 1 RESULTS f1 orl Assay for Frameshifting. Into the promoter vector p375 we inserted the phage fl origin, a translational start site, and, FIG. 1. (A) Gene organization of the L-A (+)-strand (from ref. downstream ofthe multiple cloning site, the lacZ gene in each 26). ORF1 encodes the major coat protein. ORF2 overlaps with of the three possible reading frames (pTI24, -25, and -26). In ORF1 by 130 nucleotides and is expressed as a fusion protein, the wild-type yeast cells, only pTI25, with ,-gal in the 0 frame 180-kDa minor coat protein. The ORF2 domain has ssRNA-binding with respect to the AUG, resulted in significant production of activity and an amino acid sequence diagnostic of viral RNA- p-gal (Fig.
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