Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5'-leader

The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.     

Cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4G dependent

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.     

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.     

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.     

SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.     

Base-pairing between untranslated regions facilitates translation of uncapped, nonpolyadenylated viral RNA

Translationally competent mRNAs form a closed loop via interaction of initiation factors with the 5' cap and poly(A) tail. However, many viral mRNAs lack a cap and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop by direct base-pairing (kissing) of a stem loop in the 3' untranslated region (UTR) with a stem loop in the 5' UTR. This allows a sequence in the 3' UTR to confer translation initiation at the 5'-proximal AUG. This base-pairing is also required for replication. Unlike other cap-independent translation mechanisms, the ribosome enters at the 5' end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by which mRNA UTRs can communicate.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes

The 5′-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5′-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5′-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5′-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)⁺ but not poly(A)^– mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia

Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.     

The 3'' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.     

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5'' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

A viral sequence in the 3''-untranslated region mimics a 5'' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.     

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.     

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.