Vol. 66, No. 4

JOURNAL OF VIROLOGY, Apr. 1992, p. 1924-1932

0022-538X/92/041924-09$02.00/0 Copyright © 1992, American Society for Microbiology

The Sequence Context of the Initiation Codon in the Encephalomyocarditis Virus Leader Modulates Efficiency of Internal Translation Initiation MONIQUE V. DAVIES AND RANDAL J. KAUFMAN* Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140-2387 Received 6 September 1991/Accepted 19 December 1991

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a capindependent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-li. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.

Picornavirus mRNA translation initiates through direct, cap-independent internal binding of ribosomes mediated by a complex interaction between at least two distinct regions in the 5' untranslated regions (UTRs) of these mRNAs which may involve specific cellular proteins (9, 13, 28, 29, 33). On the basis of sequence homology between putative internal ribosomal entry sites (IRES) within their 5' UTRs, the picornaviruses have been classified into three groups: (i) hepatitis A virus, (ii) the cardioviruses and aphthoviruses, and (iii) the enteroviruses and rhinoviruses. Cardiovirus and aphthovirus mRNAs, as represented by encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus, are translated with very high efficiency and accuracy in the rabbit reticulocyte lysate, while poliovirus and human rhinovirus mRNAs are translated inefficiently and inaccurately in the rabbit reticulocyte lysate but efficiently and accurately in HeLa cell extracts (5, 33). The IRES nucleotide sequence is conserved within each class, but little conservation is found between the different classes. For cardioviruses, enteroviruses, and rhinoviruses, two domains within the 5' UTR are essential for internal initiation: a stem-and-loop structure and a pyrimidine stretch in the vicinity of the initiation AUG (9, 12, 13, 33). The selection mechanism for the initiation AUG, however, may be different for each class. In poliovirus, the ribosomes bind to the IRES and then scan from approximately nucleotide (nt) 600 to the initiation codon at nt 743. This mechanism is supported by a deletion (from nt 600 to 727) which has no effect on viral infectivity (25). Furthermore, insertion of a 72-nt sequence which lacks AUG codons into the 5' UTR of poliovirus at nt 702 has no effect on infectivity, but insertion of a sequence harboring an

*

AUG codon with an unfavorable sequence context for initiation produces a small-plaque phenotype (24). In contrast, studies on cardioviruses suggest that ribosomes initiate directly at AUG-11 without prior scanning (8, 14). Initiation at AUG-10 was not detected upon introduction of a frameshift mutation to allow the detection of polypeptides initiated at AUG-10. Aphthovirus mRNAs may initiate at either one of two in-frame AUGs which are 84 nt apart and are each preceded by two conserved pyrimidine stretches of approximately 20 bases (35). If the first AUG codon is in an unfavorable context for initiation (20, 21), it is very rarely used in vivo and a sizable portion of the ribosomes scan to the next AUG codon which has a favorable context in all strains. In some serotypes, both AUGs are in a favorable context for initiation and both are used in vivo and in vitro (26, 35). To study the mechanism of initiation on EMCV mRNA in vivo, we introduced the EMCV leader from nt 260 to 834 into a eukaryotic expression vector directly preceding the marker dihydrofolate reductase (DHFR) gene so that EMCV AUG-11 was either directly fused or in frame with a downstream DHFR initiation codon. The efficiency of use of EMCV AUG-11 as well as the occurrence of initiation at downstream AUGs was assessed by transient expression in COS-1 monkey cells transfected with the various constructs and analysis of labeled cell extracts by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). We have evaluated the effect of specific base substitutions around AUG-10 and AUG-11 which affect utilization of AUG-11 as well as initiation at downstream AUG codons. The results show significant initiation at codons downstream of AUG-11, and the efficiency of AUG-11 utilization is increased following nucleotide

Corresponding author. 1924

VOL. 66, 1992

changes which conserve the consensus sequence for the AUG initiation codon. MATERIALS AND METHODS Plasmid constructions. pMT21, a derivative of pMT2 (17), and pED4 have been previously described (18). Changes in the EMCV leader region (nt 828 to 846) were introduced by using oligonucleotide-directed mutagenesis by the gapped heteroduplex procedure (30), with modifications as described previously (17) to yield the vectors pEDn (pED1 to pED5). All mutations were confirmed by DNA sequence analysis (36). A 1.6-kb eIF-2ao cDNA was obtained by digestion of peIF-2a VA+ (17) with EcoRI and ligated into the EcoRI site of pMT21 and pEDn, to yield dicistronic vectors pMT21-2cx and pEDn-2a. DNA transfections and analysis. COS-1 monkey cells were transfected by the DEAE-dextran procedure, with the addition of a chloroquine treatment as described previously (16). After 42 h, cells were labeled with [35S]methionine (100 ,uCi/ml; 1,000 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) for 30 min in methionine-free minimal essential medium. Cell extracts were prepared by lysis in Nonidet P-40 as described previously (17) and analyzed by reducing SDS-PAGE (27), either before or after immunoprecipitation with rabbit polyclonal anti-mouse DHFR kindly provided by Joseph Bertino (Sloan-Kettering Memorial Cancer Institute, New York, N.Y.). Gels were fixed in 40% methanol-10% acetic acid, prepared for fluorography by treatment with En3Hance (New England Nuclear Corp.), and dried. Dried gels were autoradiographed with Kodak XAR-5 film with a Dupont Cronex Lightning-Plus screen. Protein levels were estimated by visual comparison of band intensities from multiple autoradiograms of different exposure times. Total RNA was prepared by guanidine thiocyanate extraction (3) and analyzed by Northern (RNA) blot hybridization (38) following electrophoresis on formaldehyde-formamide denaturing agarose gels as described previously (4). Hybridization was carried out by using a DHFR probe prepared by [32P]dCTP labeling, using random priming with oligonucleotides as described by the supplier (Pharmacia Inc.). Electroporation and selection of DHFR-deficient CHO cells (DUKX-B11) was performed as described previously (39), with modifications. Subconfluent cultures of cells were trypsinized, and 2 x 106 cells were resuspended in 0.9 ml of Dulbecco's minimal essential medium supplemented with 10 mM glutamine. DNA (100 ,ug) was linearized by digestion with NdeI, resuspended in 0.1 ml of sterile water, and added to the cells. This mixture was exposed to 200 V at 1,250 ,uF (Cell ZapII; Andersen Electronics, Brookline, Mass.), and the cells were plated within 5 min into nonselective medium. Two days later, cells were subcultured 1:15 either into selective medium lacking the nucleosides or into the same medium containing increasing amounts of methotrexate (MTX). After 10 days to 2 weeks, colonies were counted after staining with methylene blue.

RESULTS Construction of expression vectors incorporating the EMCV leader. To determine the sequence requirements around AUG-11 for efficient translation initiation mediated by the EMCV leader, the EMCV leader sequence (nt 260 to 834) was inserted upstream of the reporter gene DHFR in the vector pMT21 (18). To show that internal initiation mediated by the EMCV leader is independent of the expression of an

EMCV TRANSLATION INITIATION

1925

upstream open reading frame, dicistronic vectors were constructed by inserting a 1.6-kb eIF-2a cDNA into the unique EcoRI site preceding the EMCV leader. Schematic diagrams of monocistronic and dicistronic constructs used in this study are shown in Fig. 1A. Changes were introduced around the initiation sites by using gapped heteroduplex mutagenesis methods, generating the vectors pEDn. Sequence junctions between the EMCV leader and DHFR coding regions in the different constructs are shown in Fig. 1B. Utilization of EMCV AUG-11 and downstream initiation codons. The efficiency of use of the authentic EMCV initiation codon AUG-11 (designated *11) at nt 834 and the occurrence of initiation at downstream AUG codons was monitored by derivation of pED1, pED2, and pED3 (Fig. 1B). pEDi fuses *11 with the authentic DHFR initiation codon (designated Dl), resulting in a common initiation codon (*11/Dl). pED2 places Dl in frame with *11 by inserting a 12-bp oligonucleotide containing a XhoI site between *11 and Dl. pED3 retains the EMCV leader sequence up to EMCV AUG-12 (designated *12), which is fused to Dl, resulting in a common initiation codon (*12/Dl). pMT21, which encodes the authentic murine DHFR cDNA and does not contain the EMCV leader, was used as a control. Expression of DHFR from these four constructs was analyzed by transient transfection of COS-1 monkey kidney cells. EMCV vectors were transfected in duplicate, using independently derived clones from heteroduplex mutagenesis. DHFR synthesis was studied by [35S]methionine pulse-labeling of transfected cells and analysis of the total cell extracts by SDS-PAGE (Fig. 2A). Parallel plates of transfected cells were used to isolate total cellular RNA, which was analyzed by Northern blot hybridization to a DHFR-specific probe (Fig. 2B). In total cell extracts prepared from cells transfected with pMT21 (Fig. 2A, lane 2) as well as in cells transfected with pED1 (Fig. 2A, lanes 3 and 4), a major 21.5-kDa species which corresponds to DHFR initiated at Dl was detected. Extracts prepared from cells transfected with pED2 (Fig. 2A, lanes 5 and 6) and pED3 (Fig. 2A; lanes 7 and 8) showed, in addition to DHFR, a slightly more slowly migrating species. The slower mobility is consistent with a DHFR species containing an additional five (Fig. 2A, lanes 5 and 6) or four (Fig. 2A, lanes 7 and 8) amino acids corresponding to initiation at *11. In all three vectors, the major site of initiation was *11, although in pED2 and pED3 a considerable amount of initiation also occurred at the downstream AUG, Dl. Initiation at both *11 and Dl was approximately fourfold greater in pED3 (Fig. 2A, lanes 7 and 8) than in pED2 (Fig. 2A, lanes 5 and 6). This finding suggests that nucleotide sequences 3' to the EMCV AUG-11 initiation codon can influence initiation at EMCV AUG-11. Northern blot analysis of RNA harvested from parallel transfected plates revealed similar levels of a single mRNA species of the expected size (Fig. 2B). Therefore, DHFR synthesis in these transfected cells reflected translation efficiency. Alteration of the sequence context of EMCV AUG-11 reduces its efficiency and increases downstream initiation. A purine (usually A) in position -3 of the initiation codon is the most conserved nucleotide in all eukaryotic mRNAs (20). In the literature, either an A (13, 14) or a U (31) was reported at the -3 position (residue 831) with respect to *11. To evaluate the importance of this residue, the A at position -3 (present in vectors pED1, pED2, and pED3) was mutated to T by site-directed mutagenesis in pED1 and pED2 (Fig.

1926

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DAVIES AND KAUFMAN

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FIG. 1. (A) Schematic diagrams of vectors used in this study. The monocistronic vector pMT21 contains the simian virus 40 (SV40) origin of replication and enhancer element (HindIII-PvuII fragment), the adenovirus major late promoter (AdMLP) from the XhoI site (15.83 map units) to the 5' cap site, the tripartite leader (TPL; 180 bp of the first two and two-thirds of the third leader from adenovirus major late mRNAs), an intervening sequence composed of the 5' splice site from the first leader of adenovirus major late mRNAs and a 3' splice site from an immunoglobulin gene (IVS), three unique cloning sites (PstI, EcoRI, and XhoI), a murine DHFR coding region (DHFR), the simian virus 40 early polyadenylation signal (SV40polyA), and the adenovirus VAI gene from HpaI (28.02 map units) to BalI (29.62 map units) (VAI). The backbone of this vector is pUC18 containing the ,-lactamase gene and the Escherichia coli origin of replication. pEDn vectors are derived from pMT21 by insertion of the EMCV leader (from nt 260 to 834) upstream of DHFR in the unique cloning sites EcoRI and XhoI. Changes were introduced around the EMCV initiation codon by using heteroduplex mutagenesis methods. For dicistronic vectors, eIF-2a cDNA, a 1.6-kb fragment excised from peIF-2a VA+ (17), was inserted into the EcoRI site of pMT21 and pEDn to yield pMT21-2a and pEDn-2a. In the vectors pEDn-2a, the EMCV leader is positioned between the eIF-2a and DHFR genes, while in pMT21-2a, the DHFR gene is directly adjacent to eIF-2a. (B) Sequence junctions between the EMCV leader and DHFR cDNA. The EMCV leader sequence is highlighted by a thick black line above the sequence. The EMCV AUGs are underlined and labeled *10, *11, and *12. The authentic DHFR initiation codon is underlined and labeled Dl. The second DHFR AUG is also underlined and labeled D2. When two AUGs are fused, the common AUG is underlined and labeled *11/D1 for the fusion of EMCV AUG-11 and DHFR AUG-1 or *12/Di for the fusion of EMCV AUG-12 and DHFR AUG-1. The XhoI linker sequence is underlined by a thin line. The mutation of A to T is indicated by a bold letter in pED1 A-*T and pED2 A--T. The TGA stop codon in frame with EMCV AUG-10 is underlined in pED1 A--T and changed to a TGG in pED1 A-*TAS. The sequence for the DHFR cDNA is identical in all vectors. The control expression vector pMT21 does not contain the EMCV leader.

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EMCV TRANSLATION INITIATION

VOL. 66, 1992

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FIG. 2. Utilization of EMCV AUG-11 and downstream initiation codons. The indicated plasmids were transfected into COS-1 monkey cells. pED1, pED2, and pED3 were transfected in duplicate. (A) At 42 h posttransfection, cells were pulse-labeled for 30 min with [35S]methionine and total cell extracts prepared for analysis by SDS-PAGE as described in Materials and Methods. Equivalents amount of cell extracts were loaded per lane (on the basis of trichloroacetic-precipitable counts per minute). Migration of DHFR is indicated by an arrow. All DHFR species are labeled according to their initiation codon. Molecular weight markers (in kilodaltons) are indicated on the left. (B) From parallel plates, at 42 h posttransfection, total cell RNA was harvested and analyzed by Northern blot hybridization to a DHFR probe as described in Materials and Methods.

1B). The relative use of *11 and the downstream AUG in all four constructs was tested by transient transfection in COS-1 cells (Fig. 3A). The utilization of *11 fused to Dl (*11/Dl) in pED1 A-*T (Fig. 3A, lane 5) was approximately fourfold lower than in pED1 (Fig. 3A, lane 3). In addition, a new polypeptide of 19.5 kDa was detected. The migration of this new polypeptide species corresponds to initiation at DHFR AUG-2 (D2), 42 nt downstream from Dl. Identification of this new polypeptide as DHFR was confirmed by immunoprecipitation with a polyclonal rabbit anti-DHFR (Fig. 4B, lane 10). Initiation at D2 was also detected from pED1, although at a lower level (Fig. SA, lanes 14 and 24). The utilization of *11 in pED2 A- T (Fig. 3A, lane 6) was also decreased relative to the use of *11 in pED2 (compare bands *11 in Fig. 3A, lanes 4 and 6), while the use of the next downstream AUG, Dl, was increased. In addition, whereas utilization of D2 could be detected in pD2 A--T (Fig. 5B, lane 21), it was considerably less than that of pED1 A-*T (Fig. SB, lane 20). These results are consistent with a scanning hypothesis in which the presence of an upstream AUG in a favorable context reduces initiation at downstream AUG codons. mRNA levels from all plasmids were similar, indicating that differences in DHFR synthesis resulted from

7

FIG. 3. Evidence that alteration of the sequence context of EMCV AUG-11 reduces its efficiency and increases downstream initiation. (A) The indicated plasmids were transfected into COS-1 cells, and equivalent amounts of cell extracts (on the basis of trichloroacetic acid-precipitable counts per minute) were analyzed as described for Fig. 2. Migration of DHFR is indicated by an arrow. Initiation codons for the various DHFR species are identified for each plasmid. (B) From parallel plates, total cell RNA was isolated and analyzed by Northern blot hybridization to a DHFR probe.

translation efficiency of the mRNAs (Fig. 3B). These results indicate that the adenine at position -3 of *11, which meets the consensus sequence requirement for efficient initiation, enhances initiation at *11. Mutation of -3 A to T resulted in a greater percentage of ribosomes bypassing AUG-11 to initiate at a downstream AUG having a more favorable context (Fig. 1B). EMCV AUG-10 is not required for internal initiation. DHFR translation from pED1 A-*T yielded detectable initiation at D2 (Fig. 3A, lane 5; Fig. 5A, lane 6). The sequence between EMCV AUG-10 (*10) and D2 contains a stop codon TGA in frame with *10 but not with *11/D1. To determine whether the initiation at D2 resulted from initiation at *10, termination, and reinitiation, the stop codon TGA in pED1 A-*T was mutated to a TGG in pED1 A-*TAS. Cells transfected with pED1 A-*T (Fig. 4A, lane 3) and pED1 A-*TAS (Fig. 4A, lane 4) exhibited similar levels of DHFR polypeptide initiation at D2. Therefore, mutation of the stop codon in frame with *10 did not alter initiation at D2. This result indicates that initiation at D2 did not result from initiation at AUG-10, termination, and reinitiation but more likely resulted from ribosomes bypassing *11. This conclusion is consistent with conclusions drawn from in vitro experiments showing that AUG-10 is not required for initiation (14).

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The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal...
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