Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 174-178, January 1991 Biochemistry A -1 in a double-stranded RNA virus of yeast forms a gag-pol fusion (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 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, ; ,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 27, 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 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. 3). Fragments inserted between the AUG and lacZ The encapsidation signal (27) is pres- dependent RNA polymerases. were tested for fusion of the 0 frame and the lacZ frame by ent on the (+)-strand 400 bases away from the 3' end. Overlapping with the encapsidation signal is the internal replication enhancer measurement of p-gal activity. Our evidence that this fusion (IRE) that is necessary for full template activity of (+)-strands. (B) of reading frames is due to ribosomal frameshifting depends The essential elements of the L-A region determining -1 ribosomal on detailed analysis of the region responsible for fusion ofthe frameshifting. The slippery site is the heptamer G GGU UUA (bases reading frames and comparison of this with other systems. 1958-1964). The ORF1 (gag) frame is shown by the I.I symbols on Determination of the Region of L-A Responsible for Fusing the upper row and the ORF2 (pol) frame is similarly shown in the ORF1 and ORF2. A 218-bp region from L-A (bases 1905- lower row. The two tRNAs bound on the ribosomes to the GGU 2122) including the 130-bp region of overlap of ORF1 and UUA on the mRNA slip back 1 base each to bind to GGG UUU. The ORF2 was inserted into pTI24, pTI25, and pTI26 to make stem-loop pseudoknot structure just 3' to the slippery site (bases pF7, pF8, and pF9 (Figs. 1C and 2). pF8, with p-gal in the -1 1969-2004) is also shown. A pseudoknot is a stem-loop structure to of the activity whose loop (bases with spikes) can base pair to a sequence 3' to the frame with respect the AUG, showed 1.8% base of the stem (bases with spikes). (C) Structure of the frameshift ofpTI25 (0 frame, no insert control) (Fig. 3). Because the L-A detection vector pF'8. Transcripts from the PGKI promoter are sequences inserted have stop codons in the other frames, pF7 translated from the synthetic AUG. proceeds into the (+1 frame) and pF9 (0 frame) have no significant activity. ORF1 frame of L-A. Only shifted ribosomes proceed into LacZ. The The minimum region of L-A necessary for frameshifting detailed sequence of pF'8 from the PGK promoter to the beginning was delimited by making deletions of pF'8. Deletion of 5 of LacZ is shown in Fig. 2. (pF'8-5D5), 11 (pF'8-5D11), or 17 (pF'8-5D17) codons of L-A sequence 5' to the slippery site did not diminish frameshifting in the -1 frame with respect to ORFi), pTI25 (0 reading efficiency. The deletion in pF'8-5D17 removed all 5' L-A frame), and pTI26 (+1 reading frame). pTI24, pTI25, and sequence to within 2 bases of the slippery site (Figs. 2 and 3). pTI26 were digested with BamHI and Sma I and a 218-bp Elimination of 32 codons of L-A sequence from the 3' end of Sau3a/Sma I fragment from pTIL121, containing the entire the insert had no effect on frameshifting, but deletion of 38 region of overlap of ORF1 and ORF2, were cloned into codons, removing the 3' pseudoknot region, reduced 8-gal pTI24-26 to produce pF7, pF8, and pF9, respectively (see activity 20-fold (Figs. 2 and 3). A double-deletion mutant, Figs. 1C and 2). In pF7, pF8, and pF9, lacZ is in the +1, -1, pF'8-5D17/3D32XS, eliminating 17 and 32 codons from the 5' and 0 frame, respectively, relative to the AUG codon. pF8 was and 3' ends, respectively, showed only a minor effect on cleaved with Sal I and a 200-bp Sal I fragment from pDM1 (38) activity (Fig. 3). Thus, the region from the slippery site to the containing the fl origin of replication was inserted to create 3' pseudoknot is sufficient for frameshifting. pF'8 for in vitro mutagenesis. This did not affect the efficiency Structural Requirements for Frameshifting. The sequence of frameshifting (Fig. 3). requirements of the slippery site heptamer were examined Modification of pF'8. pF'8 was modified by using synthetic (Fig. 4). Changes in the first triplet (comprising a codon in the oligonucleotides and all mutations were confirmed by se- -1 frame, but parts of 2 codons in the 0 frame) that disrupt quence analysis. The pF'8-5D mutants had in-frame deletions the identity of the three bases (G GG -> A GG or G GG -- Downloaded by guest on September 27, 2021 176 Biochemistry: Dinman et al. Proc. Natl. Acad. Sci. USA 88 (1991) PGK Dromoter---> aaggaagtaa ttatctactt tttacaacaa atcta gaattc atacaaa 'Start of transcript in this direction --> < deletedin515 >l deletedin5D1I >l deletedin5D17 >l I--L-A sequence--> -1 atg act tct agg ATCAATGCGGGCGAACTTAAGAACTACTGGGGTAGTGTGCGTCGTACTCAGC M T S R I N A G E L K N Y W G S VRR T Q Q 0 frame Translation Start * C A S Y S A -1 frame slippery 5'stem 5'Pseudoknot 3'stem 3'Pseudoknot site +1 +1 region +1 < deleted in 3Da region AGGGTTTAGGAGTGGTAGGTCTTACGATGCCAGCTGTAATGCCTACCGGAGAACCTACAGCTGGCGCTG . -1 --- > ---- > < ---- < ______- G LG V V GL T M P AV M P TG E P T A G AA 0 frame G F R S G R S Y D A S C N A Y R R T Y S W R C -1 frame

< deleted in 3D32 CCCACGAAGAGTTGAULGAACAGGC +1 +1 +1 0 .0 H E E L I E Q A D N V L V E * 0 frame ends P R R V D R T G G Q C F S R V N V I E P S HG -1 frame ACCCCGCCCTACAAGGTACATACTGCAG cccggg ggt acc gat ccc gtc gtt tta caa cgt P R P T R Y I L Q SmaI D P V .....1acZ in the +1 frame in pF7 in the -1 frame in pF8 pF7 & pF9 have in the 0 frame in pF9 1 less & 1 more G here, respectively

FIG. 2. Partial sequence of the frameshift assay vector pF'8. L-A sequences start at base 1905 and end at base 2122 of the L-A sequence. The end points of the deletions used to determine the extent of the L-A sequences necessary to promote frameshifting are also indicated. The L-A sequence is in uppercase letters and the vector sequences are in lowercase letters. G AA) each substantially reduces frameshifting, as predicted pJD16, but this was still only 20o of pF'8 activity. Mutant by the simultaneous slippage model. Making both of these pJD27, with A AAC in place of U UUA, retained wild-type changes at once to produce A AA leaves frameshifting ability activity, indicating that the C in the 7th position was probably intact. These residues are thus important for frameshifting, not responsible for the decrease in frameshifting activity. and their being identical is important. Substitution of any Likewise, GGG in this position did not frameshift (pJD28). To identical 3 nucleotides in the first triplet (G GG -* A AA, G avoid potential stacking problems due to long stretches of G GG -* C CC, or G GG-+ U UU) resulted in efficient residues, the wild-type heptamer (G GGU UUA) was frameshifting. Interestingly, substitution of pyrimidines for changed to U UUG GGC in pJD28. purines in this position gave significantly more frameshifting Making the seventh position of the slippery site U (pJD29) than wild type (2.6 times for C CC and 6.7 times for U UU) or C (pJD27) gave frameshifting with equal or greater effi- (Fig. 4). Also, although background levels of frameshifting ciency than wild type (A), but a G in this position, as in pJD30 were seen in pJD11 (G GG -* A GG), pJD12 (G GG -G G AA) (G GGU UUG) or pJD36 (G GGA AAG), gave only 18% or gave 35% ofwild-type activity (Fig. 4). This may be explained 8%, respectively, of pF'8 activity. by U-G codon/anticodon base pairing in the first position of The results obtained with pF'8-3D32 and pF'8-3D38 sug- the codon in the shifted frame. gested the requirement for sequence information present in The sequence requirements of the second triplet (U UU) the former but lacking in the latter mutant. A stretch of 6 were more stringent. Only triplets of A and U yielded nucleotides in this area (GCUGGC = 3'Psi) can form a substantial levels of 8-gal activity, and identity of the three pseudoknot with 6 nucleotides in the loop of the stem-loop bases was required (Fig. 4). To examine why pJD16 (C CC) structure (GCCAGC = 5'Psi) (Fig. 2). pJD18 contained the did not frameshift efficiently, the 7th base was also changed complement of 5'Psi (GCCAGC -+ CGGUCG) and pJD19 to C (A -* C; pJD26). In this construct, the shifted tRNA in carried the complement of 3' Psi (GCUGGC -) CGACCG), the ribosomal A site would not have its wobble base mis- each thus disrupting the potential for pseudoknot formation. paired. A 2-fold increase of 8-gal activity was observed over Each showed a 20-fold decrease in 8-gal activity. pJDRPsi < .------L-A sequences------>1 ATG------GGGTTTA---5'Psi---3'Psi------LacZ --ORF1 > LAdL FRAKE ACTIVITY LI".i -O ORF2 > of^ LacZ %of %bf PLASFlID rel to AUG 0 frame pF'8 pTI244------1 < 0.01 < 0.5 pTI255---- G--- 0 100 5500 pTI2(6---- GG--- +1 < 0.03 < 2 FIG. 3. Delimitation of region necessary for frameshifting. The pF7 ._____ +1 0.01 0 5 wild-type strain 2907 was trans- pF8 (wt)- .-G--- -1 1.8 100 formed with the plasmids shown pF9 -GG--- .-G--- -1 1.91 1005 and midlogarithmic phase cells pF'8 (wt) were assayed for 8-gal activity. pF'8- The main features of the L-A se- 5D5 --- -1 1.6 90 quence and the reading frame be- 5D11 --- -1 3.0 170 fore and after the shift are indi- 5D17 --- -1 2.4 130 cated. Psi, psuedoknot region; % of 0 frame, actual efficiency of 3D38 ------10. 21 170 of 3D32 ------1 3. frameshifting; % pF'8, compar- 5D17/3D32XS ison to the wild-type level of ___ --- -1 1.3 72 frameshifting. Downloaded by guest on September 27, 2021 Biochemistry: Dinman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 177

.<---- L-A sequences------>1 ATG------GGGTTTA----5'Psi----3'Psi --ORF1 > " U ACTIVITY | @2 _0RF2- %c)f %of PLASMIDS (from pF'8) 0 fr:ame pF'8 pF'8 (wt)------1. 91 100 pJD11------AGG------0.,03 1 pJD12------GAA------0.,4 21 pJD13------AAA------2.,2 120 pJD31------CCC------4. 7 261 pJD32------TTT------12. 0 667 pJD22------ATT------0. 11 6 pJD23------TAA------0. 19 10 FIG. 4. Effect on frameshifting pJD24------AAA------5. 0 270 of alterations in the slippery site pJD14------CTT------0. 09 5 and the pseudoknot. For alter- pJD15------TCC------0. 05 3 ations ofthe slippery site, all bases pJD16------CCC------0. 17 9 of pF'8 that are not shown were pJD26------CCCC------0. 41 20 left unchanged. pJD18 has, in the pJD27------AAAC------3. 45 150 loop of the stem-loop, the change pJD28------TTTGGGC------0. 11 6 pJD29------T------4. 1 (GCCAGC -* CGGUCG), dis- pJD30------G------O.:33 228 rupting the pseudoknot, and pJD36------AAAG------0.,16 8 pJD19 has the change (GCUGGC -* CGACCG) 3' of the stem with pJD18------c5'Psi------0.05 3 the same result. pJDRPsi has both pJD19------c3'Psi- 0.10 5 changes so the pseudoknot can pJDRPsi------c5'Psi---c3'Psi- 1.91 100 again form. contained the mutations at both sites restoring both the nonwobble bases, some frameshifting was observed. In potential for pseudoknot formation and the a-gal activity. pJD12 (G AAU UUA), the peptidyl-tRNA reading AAU Thus, the ability to form a pseudoknot structure was neces- shifts to read GAA, with pairing of G on the mRNA with U sary for efficient frameshifting. on the tRNA. Although frameshifting is decreased 4-fold, this The pseudoknot structure (Figs. 1B and 2) could be ex- is still an order of magnitude above background. This G-U tended by 4 bases (UGUA = 5' and UACA = 3') by pairing in a nonwobble position of the codon is also known to unwinding the upper portion of the stem-loop. This may be occur in the suppression of amber codons by tRNA fn6 (40, slightly favored because a G-U base pair (1 H bond) at the top 41). The pro-pol overlap of murine mammary tumor virus ofthe stem would be replaced by an A-U base pair (2 H bonds) contains a G GAU UUA heptamer (see ref. 4) in which G-U in the extended pseudoknot. In pJD20 a C replaced the native pairing is necessary in the second (nonwobble) position to U, favoring the stem. 8-Gal assays of this mutant showed fulfill the requirements of the model. only slightly higher frameshifting activity (170%o of control). In the second triplet, only A AA and U UU gave efficient The stem also contains a COU bulge (Figs. 1C and 2), frameshifting; G GG and C CC did not. This is consistent with replaced in pJD21 with a C-G pair. This stabilization of the the known occurrence in retroviruses of only these two stem resulted in a 2.5-fold increase in frameshifting effi- triplets. That this was not due to the reluctance of the tRNA ciency. The double mutant (pJD17), incorporating both mu- reading CCA to read CCC (i.e., to have its wobble base tations in pJD20 and pJD21, was, unexpectedly, normal (97% mispaired) is suggested by our finding that making the of control). seventh base C to give C CCC did not substantially improve frameshifting. Jacks et al. (4) also showed that G GGG gave DISCUSSION little or no frameshifting. These results suggest that the higher The L-A ORF1/ORF2 overlap region contains sufficient energy required to unpair the tRNA properly paired to CCX information to promote fusion of ORFi and ORF2 in vivo at or GGX makes frameshifting in these cases inefficient, and a rate consistent with the observed ratio of fusion protein to not due to a reluctance to repair in the shifted (-1) frame. major coat protein. A 72-nucleotide stretch within this region Thus, the requirements at the ribosomal A site are apparently is necessary and sufficient for this process. It contains the much more stringent than those operating at the P site. Within structures predicted by the simultaneous slippage model of the slippery site, one limiting factor in -1 ribosomal frame- Jacks and Varmus (8) to be necessary for -1 ribosomal shifting is the probability that a properly paired aminoacyl- frameshifting including the slippery site G GGU UUA and the tRNA will unpair from its 0-frame codon. ability to form an mRNA pseudoknot structure downstream The 7th base was constrained to A, U, or C. Having a G of the slippery sequence. We show that both features are residue in this position (G GGU UUG or G GGA AAG) necessary in the case of L-A for fusion of ORFi and ORF2. prevented frameshifting for reasons that are not yet clear. We conclude that the L-A dsRNA virus uses -1 ribosomal Substituting G in this position of the RSV heptamer (A AAU frameshifting to make a 180-kDa fusion protein whose N-ter- UUG) did not affect frameshifting (4). minal (gag) domain is a major coat protein monomer and tRNAs and Frameshifting. Bjork et al. (42) have shown that whose C-terminal domain is a ssRNA-binding region with the 1-methylguanosine in position 37 (next to the anticodon) of consensus sequence typical of RNA-dependent RNA poly- tRNAPr° prevents frameshifting, in this case the suppression merases. in Salmonella ofa frameshift mutation. Hatfield and Oroszlan Several aspects of our results, and those of others working (6) suggest that hypomodification of bases in the anticodon on retroviruses, are not explained by the Jacks and Varmus loop of the tRNAs in and around the shift site gives greater model. In the first triplet, any 3 identical bases were sufficient flexibility of movement (including slipping) of the tRNA in to yield efficient frameshifting. We show that, in addition to the mRNA/ribosome context. Indeed, infection with several the known occurrence of G GG, A AA, and U UU in this retroviruses may be accompanied by hypomodification of position, C CC is also consistent with efficient frameshifting. just those tRNAs that would be at the A site of frameshifting Furthermore, as long as base pairing was possible in the ribosomes (43). Downloaded by guest on September 27, 2021 178 Biochemistry: Dinman et al. Proc. Natl. Acad. Sci. USA 88 (1991) Examination of the available sequences (44) of P-site or of 2. Shinnick, T. M., Lerner, R. A. & Sutcliffe, J. G. (1981) Nature A-site anticodons that, in our experiments, do and do not (London) 293, 543-548. promote frameshifting does not reveal any significant differ- 3. Yoshinaka, Y., Katoh, I., Copeland, T. D. & Oroszlan, S. (1985) Proc. Natl. Acad. Sci. USA 82, 1618-1622. ences in the degree of modifications seen in the anticodon 4. Jacks, T., Madhani, H. D., Masiarz, F. R. & Varmus, H. E. (1988) loops of the two classes oftRNAs. For example, we find that Cell 55, 447-458. the tRNALYS decoding AAA is capable of efficient frame- 5. Hatfield, D. L., Lee, B. J., Feng, Y. X., Levin, J. G., Rein, A. & shifting (pJD24; Fig. 4), showing that A-site "shifty" tRNAs Oroszlan, S. (1989) Proteases ofRetroviruses (de Gruyter, Berlin). are not limited to the naturally occurring tRNAASll decoding 6. Hatfield, D. & Oroszlan, S. (1990) Trends Biochem. Sci. 15, AAC, the tRNAPhe decoding UUU, and the tRNALCU reading 186-190. UUA. 7. Schwartz, D. E., Tizard, R. & Gilbert, W. (1983) Cell 32, 853-869. 8. Jacks, T. & Varmus, H. E. (1985) Science 230, 1237-1242. Belcourt and Farabaugh (45) propose that the + 1 frame- 9. Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J. shifting in Tyl occurs when the 0-frame A-site AGG codon is & Varmus, H. E. (1988) Nature (London) 331, 280-283. not filled because tRNAAG is scarce. The P-site 0 and +1 10. Brierley, I., Boursnell, M. E., Binns, M. M., Bilimoria, B., Blok, frame leucine codons CUU and UUA can be recognized by V. C., Brown, T. D. K. & Inglis, S. C. (1987) EMBO J. 6, 3779- the same, abundant, tRNAIeU whose anticodon is UAG. The 3785. translational pause allows this tRNALCU at the P site to slip 11. Bredenbeek, P. J., Pachuk, C. J., Noten, A. F. H., Charite, J., forward 1 base from CUU to UUA. As applied to L-A the first Luytjes, W., Weiss, S. & Spaan, W. J. M. (1990) Nucleic Acids Res. 18, 1825-1831. codon in the 0 frame after the slippery site is GGA, a rarely 12. Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477-535. used glycine codon (46). This could produce a translation 13. Blinkowa, A. L. & Walker, J. R. (1990) Nucleic Acids Res. 18, pause that could promote -1 ribosomal frameshifting. How- 1725-1729. ever, such a shift would be into the equally rare arginine AGG 14. Tsuchihashi, Z. & Kornberg, A. (1990) Proc. Natl. Acad. Sci. USA codon in wild-type L-A and into the rare arginine CGG codon 87, 2516-2520. in pJD27 (GGGAAAC). Such a mechanism is also unlikely 15. Flower, A. M. & McHenry, C. S. (1990) Proc. Natl. Acad. Sci. USA 87, 3713-3717. because it requires the ribosomes to sense the three codons 16. Clare, J. J. & Farabaugh, P. J. (1985) Proc. Natl. Acad. Sci. USA at once. If the ribosomes were waiting for the tRNA recog- 82, 2829-2833. nizing GGA to arrive at the A site, the tRNA that had 17. Mellor, J., Fulton, S. M., Dobson, M. J., Wilson, W., Kingsman, recognized GGU would have already been released, and so S. M. & Kingsman, A. J. (1985) Nature (London) 313, 243-246. the first triplet (G GG) would not be important. 18. Clare, J. J., Belcourt, M. & Farabaugh, P. J. (1988) Proc. Natl. Acad. Sci. USA 85, 6816-6820. Different viruses have different sequences within the con- 19. Craigen, W. J., Cook, R. G., Tate, W. P. & Caskey, C. T. (1985) straints of the slippery site rules perhaps because each virus Proc. Nail. Acad. Sci. USA 82, 3616-3620. has its own stoichiometric requirements for structural versus 20. Weiss, R. B., Dunn, D. M., Atkins, J. F. & Gesteland, R. F. (1987) enzymatic proteins. The L-A virion has -120 major coat Cold Spring Harbor Symp. Quant. Biol. 52, 687-693. protein molecules per viral particle. The 1.8% frequency of 21. Weiss, R. B., Dunn, D. M., Dahlberg, A. E., Atkins, J. F. & frameshifting observed here suggests that there are two Gesteland, R. F. (1988) EMBO J. 7, 1503-1507. 22. Hizi, A., Henderson, L. E., Copeland, T. D., Sowden, R. C., fusion proteins per virion. Presumably, L-As with slippery Hixson, C. V. & Oroszlan, S. (1987) Proc. Natl. Acad. Sci. USA 84, sites with different frequencies of frameshifting would either 7041-7046. be inviable due to improper virion assembly or would be 23. Brierley, I., Digard, P. & Inglis, S. (1989) Cell 57, 537-547. present in lower copy number in the infected yeast cell. 24. Pleij, C. W. A., Rietveld, K. & Bosch, L. (1985) Nucleic Acids Res. Why Do Viruses Use Frameshifting and Translational Sup- 13, 1717-1731. 25. Puglisi, J. D., Wyatt, J. R. & Tinoco, 1. (1988) Nature (London) 331, pression to Make gag-ol Fusion Proteins? Ribosomal frame- 283-286. shifting is utilized by retroviruses to make a gag-pol fusion 26. Icho, T. & Wickner, R. B. (1989) J. Biol. Chem. 264, 6716-6723. protein in a fixed ratio to the gag protein. One reason for 27. Fujimura, T., Esteban, R., Esteban, L. M. & Wickner, R. B. (1990) making a gag-pol fusion protein is probably that the gag Cell 82, 819-828. domain provides a means to anchor the reverse transcriptase 28. Sommer, S. S. & Wickner, R. B. (1982) Cell 31, 429-441. to the particle. It has also been suggested (29) that the pol 29. Fujimura, T. & Wickner, R. B. (1988) Cell 55, 663-671. 30. Wickner, R. B. (1989) FASEB J. 3, 2257-2265. domain may be involved in recognition, binding, and pack- 31. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol. aging of the genomic RNA itself, as appears to be the case in 153, 163-168. the yeast L-A dsRNA virus. Retroviruses use ribosomal 32. Wickner, R. B. (1979) Genetics 92, 803-821. frameshifting and, in some cases, translational read-through 33. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular of termination codons tojoin the 5' gag ORF to the 3' pol (or Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold pro) ORF. It has been suggested (26) that since retroviruses, Spring Harbor, NY). 34. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. dsRNA viruses, and (+)-strand RNA viruses all package and 35. Tabor, S. & Richardson, C. C. (1987) Proc. Natl. Acad. Sci. USA replicate their (+)-strands, the use of splicing or other 84, 4767-4771. modifications of mRNA molecules must be accompanied by 36. Hitzeman, R. A., Leung, D. W., Perry, L. J., Kohr, W. J., Levine, excision of packaging or replication signals to avoid the H. L. & Goeddel, D. V. (1983) Science 219, 620-625. generation ofmutant genomes. Indeed, retroviruses regularly 37. Casadaban, M. J., Martinez-Arias, A., Shapiro, S. K. & Chou, J. the and other (1983) Methods Enzymol. 100, 293-308. splice to form the env protein (and tat, rev, 38. Mead, D. A., Skorupa, E. S. & Kemper, B. (1985) Nucleic Acids minor proteins in human immunodeficiency virus). These Res. 13, 1103-1118. splicing events remove the Psi packaging site. (+)-Strand 39. Guarente, L. (1983) Methods Enzymol. 101, 181-191. RNA viruses and dsRNA viruses apparently avoid this 40. Lin, J. P., Aker, M., Sitney, K. C. & Mortimer, R. K. (1986) Gene problem by not using splicing. Using ribosomal frameshifting 49, 383-388. or nonsense codon read-through to fuse gag and pol ORFs 41. Weiss, W. A. & Friedberg, E. C. (1986) J. Mol. Biol. 192, 725-735. 42. Bjork, G. R., Wikstrom, P. M. & Bystrom, A. S. (1989) Science likewise prevents generation of mutants. 244, 986-989. 43. Hatfield, D., Feng, Y.-X., Lee, B. J., Rein, A., Levin, J. G. & The authors are grateful to Dolph Hatfield and Luz Hermida- Oroszlan, S. (1989) Virology 173, 736-742. Rodriguez for useful discussions. 44. Sprinzl, M., Hartmann, T., Weber, J., Blank, J. & Zeidler, R. (1989) Nucleic Acids Res. Suppl. 17, 1-172. 1. Weiss, R., Teich, N., Varmus, H. & Coffin, J. (1982) RNA Tumor 45. Belcourt, M. F. & Farabaugh, P. J. (1990) Cell 62, 339-352. Viruses (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 46. Ikemura, T. (1982) J. Mol. Biol. 158, 573-597. Downloaded by guest on September 27, 2021