key: cord-0009297-wqy89i4v
authors: Hatfield, Dolph; Oroszlan, Stephen
title: The where, what and how of ribosomal frameshifting in retroviral protein synthesis
date: 2003-03-21
journal: Trends Biochem Sci
DOI: 10.1016/0968-0004(90)90159-9
sha: 3e1b9ac43cdc25c5d4e296b13c41503868f9d622
doc_id: 9297
cord_uid: wqy89i4v

The gag and pol genes of most retroviruses occur in different reading frames and their translation as a single polypeptide is carried out by ribosomal frameshifting in the −1 direction. The alignment of the different reading frames occurs by overlapping reading in response to at least two signals within the RNA: one is a heptanucleotide stretch at the frameshift site and the other is a stem-loop structure which occurs just downstream of the first signal.

The gag and pol genes of most retroviruses occur in different reading frames and their translation as a single polypeptide is carried out by ribosomal frameshifting in the -1 direction. The alignment of the different reading frames occurs by overlapping reading in response to at least two signals within the RNA: one is a heptanucleotide stretch at the frameshift site and the other is a stem-loop structure which occurs just downstream of the first signal.

utilizes ribosomal frameshifting to align two large, briefly overlapping reading framesT. Previously, a popular concept was that alignment of the different reading frames occurred by a small splice in the mRNA, but this possibility was ruled out when the alignment was shown to occur by ribosomal frameshiftingT-9. In fact, we now know where the frameshift occurs. We also know a great deal about what information is present in RNA for aligning the different reading frames. In addition, we know how the different reading frames are aligned and we have some insight into how the frameshift is accomplished; i.e. how the fidelity of translation is maintained during the repositioning of the ribosome on the mRNA.

Overlapping genes occur in the gagpro-pol translational unit of the genomesized mRNA of many retroviruses. These genes are all expressed from the same AUG initiation codon. The gag gene encodes the structural proteins of the virus and the pol gene encodes the replication enzymes, reverse transcriptase and integrase. The replication enzymes are usually synthesized at a 5-10% level of the Gag proteins. The pro gene, which encodes the viral protease, can be expressed in the same reading frame as gag and pol or in a separate reading frame overlapping gag and/or pol. For example, in mammalian type C retroviruses, the pro and pol genes are expressed through suppression of the termination codon at the end of gag by a glutamine tRNA10; and thus, the same reading frame is maintained in translation of gag and pol. This means of expressing the Gag-Pol fusion protein is designated as in-frame readthrough ( Fig. 1) . In other retroviruses, the genes downstream of gag occur in different reading frames. Expression of the Gag-Pro-Poi fusion protein in some of these retroviruses requires a single frameshift event in the -1 direction to align the different reading frames, whereas expression in others requires two such events, one between gag and pro and the other between pro and pot (Fig. 1) .

The frameshift occurs within the region known as the overlapping reading frame. The boundaries of this region (also designated as the overlap or frameshift window) are established, for example, in retroviruses requiring a single frameshift event, by the termination codon at the end of gag (the 0 reading frame) and the first upstream termination codon in the -1 frame (the pol reading frame). The size of the overlap may vary from a few nucleotides (e.g. 13 [UUU] are read in the 0 frame), whose role is to signal the frameshift event,s. Mutagenesis studies within and around the frameshift region show that the signal actually consists of a heptanucleotide sequenc@,~. Examples of frameshift signals are shown in Table I .

The heptanucleotide frameshift signal may occur anywhere within the overlap from the extreme 3' end (e.g. just before the gag termination codon in RSV)~5 to near the 5' end (e.g. just inside the 5' boundary of the window in human immunodeficiency virus-1 [HIV-1] which is 234 nucleotides upstream of the gag termination codon)]7. The site of the frameshift is the 3' base at the end of the heptanucleotide signal (designated with an arrow in Fig. 2 ). This was demonstrated by sequencing the transframe protein or peptide, synthesized either in vivo8 or in vitro (Refs 15 and 18, and S. H. Nam, T. D. Copeland, M. Hatanaka and S. Oroszlan, unpublished), through the frameshift site (see Fig. 2 ). The data show, for example, that leucine and isoleucine are generated from the UUAUA sequence in RSV (where UUA is the 3' terminal codon of the frameshift signal)Is; and isoleucine is read in the -1 frame by AUA and leucine in the 0 frame by either UU (if the frames are aligned by two out of three base reading) or UUA (if the frames are aligned by overlapping reading). Similarly, asparagine and proline are generated from the AACCA sequence in the pro--pol HTLV-1 overlapping reading frame (where AAC is the 3' terminal codon of the frameshift signal); and proline is read in the -1 frame by CCA and asparagine in the 0 frame by either AA or AAC. Thus, these studies define the frameshift site, but do not demonstrate whether the different reading frames are aligned as a result of two out of three base reading (doublet decoding) or overlapping reading as originally proposed by Hizi et al. 8 . We will return to this question in a later section.

There are at least two types of information present in RNA which signal the frameshift event. One type is encoded in the heptanucleotide signal discussed above. As noted, mutagenesis studies show that only the seven nucleotide stretch is required at the frameshift sitelS,]~ for the shift to occur. This conclusion is supported by other studies in which efficient frameshifting is maintained when the codon immedi- ately upstream (in the -1 frame) and that immediately downstream (in the 0 frame) of the heptanucleotide frameshift signal in coronavirus infectious bronchitis virus 0BV) are replaced with nonsense codons (i.e. the frameshift window consists of only a seven nucleotide stretch; S. lnglis, pets. commun.). The second type of information which has an important role in frameshifting is RNA secondary structure. Stem-loop structures occur just downstream of the gag--pol and of the gag-pro and pro-pol retroviral frameshift sitesTj3,~s,~a,~9 as well as just down-stream of the frameshift site in IBV20. 21 . In RSV, disruption of base pairings within the stem by generating specific stem-destabilizing mutations results in a decrease in frameshifting, while restoring these base pairings by generating specific stem-restabilizing mutations rescues frameshifting~5. Deletion of certain bases further downstream of the stem-loop structure in IBV also inhibits frameshifting suggesting that many of these downstream bases interact with the stem-loop resulting in a tertiary structure known as a pseudoknot2L The occurrence and role of pseudoknots in retroviruses and in Fig. 3 . Location of the stem-loop structure relative to the frameshift site is also important since altering the distance between these two elements by as few as three nucleotides in either direction inhibits frameshifting2L Furthermore, the stem-loop structures are thermodynamically highly stable and their involvement in frameshifting is statistically relevant relative to other such configurations which may occur within several hundred nucleotides upstream or downstream of the frameshift site 24. It seems, therefore, that the ribosomal frameshift event requires a carefully positioned downstream stemloop structure for efficient frameshifting which may function to pause translation long enough for the shift to occur. 

Sequence of the transframe protein at the frameshift site. Underlined letters show the frameshift signals; normal letters, the 0 reading frame; letters in italics, the -1 reading frame; an arrow indicates the frameshift site and u signifies the site of the mutation.

Mutations in the 3' base at the end of the heptanucleotide signal involving UUU or UUA codons do not inhibit frameshifting]s.~6. Since the sequence of the transframe peptide shows that the shift has to occur at the codon of which this is the third base, this suggests that the alignment must occur by doublet decoding. However, as sound as this argument may seem, the sequence of a transframe peptide generated from a frameshift signal containing a mutation in the 3' base demonstrates that the alignment occurs by overlapping reading. A single base change at the 3' end of the frameshift signal results in two new amino acids in the transframe protein. That is, alteration of the RSV UUAUA sequence, which codes for leucine (UUA) in the 0 frame and isoleucine (AUA) in the -1 frame, to UUUUA (where U represents the altered base which occurs at the 3' end of the frameshift signal) results in the occurrence of phenylalanine (UUU) and leucine (UUA) in the transframe proteinls (see Fig. 2 ). This observation demonstrates that the base at the 3' end of the frameshift signal must be read twice; once in the 0 frame and once in the -1 frame 8. Thus, alignment of the different reading frames occurs by overlapping reading of the 3' nucleotide at the end of the frameshift signal (Fig. 2) .

The frameshift event involves translocation of the aminoacyl-tRNA and the peptidyl-tRNA (which are present in the ribosomal A-and P-sites, respectively) by one nucleotide in the 5' direction. We do not know precisely the means by which the frameshift is carried out, but the simultaneous-slippage model proposed by Jacks et al.,s provides the best explanation of the frameshift event at present. In this model, the peptidyl-tRNA located in the P-site and the aminoacyl-tRNA located in the A-site are proposed to slip simultaneously by one base in the 5' direction resulting in both tRNAs misreading the 3' base or reading two out of three bases in the corresponding mRNA. The slippage prepares the ribosome to read the -1 frame. In the next step, normal transfer of the growing polypeptide to the aminoacyl-tRNA and its translocation to the P-site would bring the first codon in the -1 frame to the A-site. The -1 reading is then consummated with normal decoding of the A-site and transfer of the nascent peptide to the incoming aminoacyl-tRNA.

If the slippage model is correct, we would expect that the codon: anticodon interactions of tRNAs in the ribosomal A-and P-sites would not be altered significantly by the shift from the 0 frame (the frame in which the tRNAs were decoded) to the -1 reading frame (the frame in which the tRNAs now have a new set of codons). Otherwise, the ribosome:codon:anticodon complex may be destabilized and fall apart. Within the heptanucleotide signal, the base in the 3' position of the upstream codon in the 0 frame (i.e. U, A or G) is identical to the bases in the first two positions of the downstream codon (i.e. UU, AA or GG, respectively; see columns A-C, Fig. 4) . Thus, the shift to the new reading frame maintains similar codon:anticodon interactions provided the isoacceptors in the A-and P-sites misread the base in the 3' position of the codon or read only two out of three bases. It should be noted that two out of three base reading cannot be distinguished from misreading the 3' base of the codon in many cases. There is a direct relationship between promotion of frameshifting and misreading or reading two out of three bases, since the same tRNAs are involved in both processes (i.e. tRNAs that promote frameshifting must then, after the frameshift event, misread the 3' base of the corresponding new set of codons). Interestingly, tRNAs which lack a modified base in their anticodon loop are known to promote frameshifting2s and, for that matter, misreading26.2L A recent analysis of the tRNA in HIV infected cells showed that most of the Phe-tRNA lacked the highly modified Wye base in its anticodon loop, while in bovine leukemia virus (BLV) and HTLV-I infected cells most of the Asn-tRNA lacked the highly modified Q base in its anticodon loop 28. This study showed a correlation between the occurrence of hypomodified tRNAs in retroviral infected cells and their utilization in translating codons within the respective frameshift signals. The lack of a hypermodified base in the anticodon loop of tRNA would create more space in and around the frameshift site and greater flexibility of movement of the tRNA anticodon would be expected in the absence of a highly modified base. Thus, decoding of a hypomodified tRNA at the frameshift site may be a requirement for promoting frameshifting28.

Within the frameshift signals analysed to date, one exception to the occurrence of the same base in the 3' position and first two 5' positions of the upstream codons is the presence of an Asp codon (GAU) in the pro-pol frameshift signal of MMTV. A shift to the -1 reading frame would result in Asp-tRNA decoding GGA (a glycine codon) (column D, Fig. 4 ). Thus it would seem that, following the shift to the -1 reading frame, mismatching between the middle and third positions of the codon, and the first and second positions of the corresponding Asp-tRNA anticodon are permitted provided the simultaneous-slippage model is correct.

Our knowledge of the frameshift event in vertebrate viruses has increased substantially since it was first demonstrated by Jacks and Varmus7 to be utilized in RSV as a means of aligning different reading frames. We now know that frameshifting in the -1 direction occurs by overlapping reading at the 3' terminal base within the frameshift signal. It is also apparent that a stem-loop structure, which occurs immediately downstream of the frameshift signal and which may also exist as a pseudoknot, is required for efficient frameshifting.

There are several areas in retroviral frameshifting of which further study is required. We need to know more about the status of the tRNAs involved in this process. We also need to know more about how the frameshift occurs and the reason it occurs at only a moderate to low level. We need to know more about the role of the ribosome in this process and if a specific protein may be required to bring about frameshifting. Additionally, it is important to know if ribosomal frameshifting in the +1 direction also occurs in higher eukaryotes. A central question to resolve, however, is whether frameshifting in the -1 direction is a requirement of the host cell. If frameshifting is not required by the host, then the frameshift event should provide a target for inhibiting expression of viruses utilizing this regulatory mechanism of gene expression in translation. 

Bases read at the ribosomal A-and P-sites within the frameshift signal after the shift of the reading frame. The sequences shown represent a summary of the ribosomal frameshift signals determined in vertebrate viruses (see Fig. 1 and Refs 15 and 28). They are arranged in four classes (columns A-D) depending on the codon:anticodon interaction after the frameshift event as follows: Shift from the 0 to the -1 frame results in misreading, or reading two out of three bases (see text) in both the ribosomal A-and P-sites (A), just the A-site (B), or just the P-site (C); in column D, a shift to the -1 frame results in reading only one base in the P-site and two bases in the A-site by the standard Watson-Crick base pairings. Squip~ly lines show the nascent polypeptides attached to tRNAs in the P-sites; AA, the amino acid attached to tRNA in the A-site, and the dashed line, mismatching in codon:anticodon interactions between standard Watson-Crick base pairs.

Jerusalem Convention Centre, Israel This meeting will feature symposia and colloquia (in up to eight parallel sessions) covering the following major topics: Area I: The geaome

We thank Drs Reed Wickner, Samuel Wilson and Pat Becerra for helpful suggestions and critical evaluation of the manuscript and Dr Stephen Inglis for permission to quote his unpublished data on IBV. This work was sponsored in part by NCI, DHHS under contract number NO1-CO-74101 with BRI.

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