Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon.

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.     

RNA sequence determinants of a coupled termination-reinitiation strategy for downstream open reading frame translation in Helminthosporium victoriae virus 190S and other victoriviruses (Family Totiviridae)

The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.     

Downstream sequence adjacent to AUG affects translation of chloramphenicol acetyl transferase in eukaryotic cells

The CAT gene is widely used as a reporter in eukaryotic systems because of the efficient translation of its mRNA. We report here that a sequence occurring in the CAT mRNA at +15 nucleotides from CAT AUG is essential for translation. This sequence includes a stem-loop structure, which, however, exhibits a calculated stability significantly lower than that required for a hairpin to act as an enhancer of translation in vitro. Replacement of this region with the corresponding sequence from mRNAs that are normally translated in eukaryotic systems drastically reduced translation of CAT in COS cells, although the consensus sequence around the AUG, known to be required for high-level translation initiation, was conserved. These observations may be relevant for the exploitation of the CAT reporter system for analysis of the mechanisms of translation initiation by means of fusion constructs.     

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus N and NS proteins.

The complete nucleotide sequences of the vesicular stomatitis virus (VSV) mRNA's encoding the N and NS proteins have been determined from the sequences of cDNA clones. The mRNA encoding the N protein is 1,326 nucleotides long, excluding polyadenylic acid. It contains an open reading frame for translation which extends from the 5'-proximal AUG codon to encode a protein of 422 amino acids. The N and mRNA is known to contain a major ribosome binding site at the 5'-proximal AUG codon and two other minor ribosome binding sites. These secondary sites have been located unambiguously at the second and third AUG codons in the N mRNA sequence. Translational initiation at these sites, if it in fact occurs, would result in synthesis of two small proteins in a second reading frame. The VSV and mrna encoding the NS protein is 815 nucleotides long, excluding polyadenylic acid, and encodes a protein of 222 amino acids. The predicted molecular weight of the NS protein (25,110) is approximately one-half of that predicted from the mobility of NS protein on sodium dodecyl sulfate-polyacrylamide gels. Deficiency of sodium dodecyl sulfate binding to a large negatively charged domain in the NS protein could explain this anomalous electrophoretic mobility.     

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.     

Protein similarity search under mRNA structural constraints: application to targeted selenocysteine insertion

Selenocysteine is the 21th amino acid, which occurs in all kingdoms of life. Selenocysteine is encoded by the STOP-codon UGA. For its insertion, it requires a specific mRNA sequence downstream the UGA-codon that forms a hairpin like structure (called Sec insertion sequence (SECIS)). We consider the computational problem of generating new amino acid sequences containing selenocysteine. This requires to find an mRNA sequence that is similar to the SECIS-consensus, is able to form the secondary structure required for selenocysteine insertion, and whose translation is maximally similar to the original amino acid sequence. We show that the problem can be solved in linear time when considering the hairpin-like SECIS-structure (and, more generally, when considering a structure that does not contain pseudoknots).     

The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA.

Bacteriophage T7's gene 0.3, coding for an antirestriction protein, possesses one of the strongest translation initiation regions (TIR) in E. coli. It was isolated on DNA fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene in an expression vector to control the translation of this gene's sequence. The TIR's efficiency was highly dependent on nucleotides +15 to +26 downstream of the gene's AUG. This sequence is complementary to nucleotides 1471-1482 of the 16srRNA. Similar sequences complementary to this rRNA region are present in other efficient TIRs of the E. coli genome and those of its bacteriophages. There seems to be a correlation between this sequence homology and the efficiency of the initiation signals. We propose that this region specifies a stimulatory interaction between the mRNA and 16srRNA besides the Shine-Dalgarno interaction during the translation initiation step.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

In polycistronic Q�� RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA.     

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.     

Recognition of 5'-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2

TAR, a 59 nt 5′-terminal hairpin in human immunodeficiency virus 1 (HIV-1) mRNA, binds viral Tat and several cellular proteins. We report that eukaryotic translation initiation factor 2 (eIF2) recognizes TAR. TAR and the AUG initiation codon domain, located well downstream from TAR, both contribute to the affinity of HIV-1 mRNA for eIF2. The affinity of TAR for eIF2 was insensitive to lower stem mutations that modify sequence and structure or to sequence changes throughout the remainder that leave the TAR secondary structure intact. Hence, eIF2 recognizes structure rather than sequence in TAR. The affinity for eIF2 was severely reduced by a 3 nt change that converts the single A bulge into a 7 nt internal loop. T1 footprinting showed that eIF2 protects nucleotides in the loop as well as in the strand opposite the A bulge. Thus, eIF2 recognizes the TAR loop and lower part of the sub-apical stem. Though not contiguous, these regions are brought into proximity in TAR by a bend in the helical structure induced by the UCU bulge; binding of eIF2 opens up the bulge context and apical stem. The ability to bind eIF2 suggests a function for TAR in HIV-1 mRNA translation. Indeed, the 3 nt change that reduces the affinity of TAR for eIF2 impairs the ability of reporter mRNA to compete in translation. Interaction of TAR with eIF2 thus allows HIV-1 mRNA to compete more effectively during protein synthesis.     

A possible explanation for the multiple polyadenylation sites in transcripts coding for a winged-bean leghemoglobin

Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean (Psophocarpus tetragonolobus L.) nodule library. Although identical in sequence, they each possess a different side of polyadenylation located 93–128 nucleotides downstream of two overlapping AAUAAA putative signal sequences. By analysis of the untranslated 3′ ends, a potential mRNA secondary structure can be predicted which could explain the observed polyadenylation heterogeneity. The structure is a size-variable hairpin, creating a net topological distance of 25–27 nucleotides between the canonical signal sequence and the different polyadenylation sites observed. We suggest that this type of variable secondary structure could be one among other causes that determines the apparent flexibility of plant polyadenylation. It could also confer particular properties to the mRNA in relation to stability, translation efficiency and-or nuclear export.     

Two Alternative Ways of Start Site Selection in Human Norovirus Reinitiation of Translation

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.     

Complete nucleotide sequence of the phosphoprotein of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus.

The complete nucleotide sequence of the phosphoprotein (P) gene of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus was determined. Comparison with the P gene of the Edmonston strain of measles virus (MV) revealed 44 differences of which 23 nucleotides substitutions were identical with those revealed between other SSPE viruses and MV (Cattaneo et al. (1989) Virology 173, 415-425). The consensus sequence of the G insertion site was completely conserved, whereas mRNAs with one or three non-templated G residue insertions were found in addition to the mRNA of the exact genome copy. As a result of the frameshift downstream of the site of G insertion, the cysteine-rich V protein was predicted from the one G-inserted mRNA besides the P and C proteins predicted from the genome-copied mRNA.