Regulation of gene expression at the translation level

the viral complementary RNA. The two ORFs are separated by an intergenic region. This is illus- trated for RNA4 of Tenuiviruses in Fig. 1C. Viruses using this strategy have evolved a unique mechanism to produce the sgRNAs which direct the synthesis of their proteins. Contrary to initia- tion by classical RdRps, initiation of synthesis of the mRNAs by a mechanism known as cap- snatching is primer-dependent, the primers deriv- ing from the 5 end of host mRNAs. Thus, the viral mRNAs are capped as opposed to the viral and viral complementary RNAs that are not capped. Nevertheless, as shown in vitro for Tenuiviruses, the uncapped viral RNAs can serve directly as templates in translation, and translation follows the scanning mode of initiation [14]. The mechanism whereby cap-snatching occurs among plant viruses is unknown.

4. Regulation of gene expression at the translation level

Regulation at the level of translation can occur at all steps: initiation, elongation and termination. 4 . 1 . Initiation 4 . 1 . 1 . Factors influencing initiation Both cis- and trans-acting elements concur to allow efficient initiation of translation of plant viruses. The cis-acting elements are discussed in Section 4.1.1.1, Section 4.1.1.2, Section 4.1.1.3, Section 4.1.1.4 and Section 4.1.1.5 and the trans- acting elements in Section 4.1.1.6 and Section 4.1.1.7. 4 . 1 . 1 . 1 . Nature of the 5 end. Initiation of protein synthesis from monocistronic mRNAs presumably generally occurs by the classical scanning mecha- nism, starting from the capped 5 end of the mRNA. In several plant virus groups, the 5 end bears a viral-coded protein virus protein genome- linked, VPg in place of a cap structure. The VPg is covalently linked to the RNA. This is the case in plant RNA viruses Como6iridae, Poty6iridae, Lu- teoviruses, Sobemoviruses that belong to the pi- corna-like supergroup rev. in Ref. [15]. By analogy to the animal Picorna6iridae, attempts have been made to determine whether in the VPg- containing plant viral RNAs, initiation of protein synthesis occurs at the level of an ‘internal ribo- some entry site’ IRES. The data indicate that in certain cases initiation possibly occurs on an inter- nal site in the 5 leader sequence even though this sequence in plant viruses is considerably shorter and presumably less structured than in animal viruses; in other cases, the data have been contro- versial rev. in Ref. [16] . Further investigations based on the use of the dicistronic mRNA assay rev. in Ref. [16] should allow conclusive results to be reached. Yet, in other plant RNA viruses such as the Necroviruses and the members of subgroup I of the Luteoviruses, the 5 terminus of the genome bears neither a cap structure nor a VPg [17] rev. in Ref. [18] . The mechanism of initiation of protein synthesis in these virus groups is discussed below. 4 . 1 . 1 . 2 . Nature of the leader sequence. Efficiency of translation can also depend on whether the 5 UTR is poorly or highly structured, and if struc- tured, where this structure resides rev. in Ref. [19]. 4 . 1 . 1 . 3 . Initiation codon. Except for two instances, an AUG codon serves as the natural initiation codon among plant viruses. ORF I of RTBV, the first large ORF following the long leader sequence with its various short ORFs, possesses an AUU initiation codon [20]. This AUU codon seems to be important for virus viability. The other case is found in Soil-borne wheat mosaic furovirus SB- WMV which contains a bipartite RNA genome. RNA2 contains the ORF for the 19-K CP. How- ever, in vitro studies with site-directed mutants have revealed that in addition to the 19-K protein, a 25-K CP-related protein is produced that is initiated at a CUG codon upstream of the AUG which initiates the 19-K protein [21]. Conservation of the CUG codon in several SBWMV isolates suggest that the 25-K protein plays a role in the virus life cycle. 4 . 1 . 1 . 4 . Context surrounding the initiation codon. A favorable context surrounding the initiation codon increases the efficiency of translation. This seems to apply to mRNAs with AUG as initiator, as well as to those with a non-AUG initiator. A purine at − 3 and a G at + 4 A of AUG is + 1 constitute a favorable context, the sequence AACCAUGG being the most favorable [22]. 4 . 1 . 1 . 5 . Influence of the 3 region. As mentioned above, the 5 terminus of the RNA can possess a cap, a VPg, or it can be devoid of either. Similarly, the 3 end can bear a polyA tail, a tRNA-like structure, or neither. An increasing number of cases is being reported in which the 3 UTR of an mRNA plays a crucial role in initiation of transla- tion of that mRNA [3]. The RNA of Tobacco etch potyvirus contains a 5 VPg and a 3 polyA tail. It has been shown that functional interaction be- tween the 5 leader and the polyA tail stimulates translation of the mRNA, possibly by way of the polyA binding protein [23]. On the other hand, the Tobamovirus genome carries a cap at its 5 end, and a tRNA-like structure preceded by an upstream pseudoknot domain at its 3 end. Here, a region within the pseudoknot domain appears to functionally re- place the polyA tail and to enhance efficiency of translation, in concert with the cap at the 5 end [24]. A similar effect has been described in the case of Brome mosaic bromovirus [25]. However, only a very minor enhancing effect of the pseudoknot upstream of the tRNA-like structure on efficiency of translation was observed with Turnip yellow mosaic tymovirus [25]. Finally, there is still another scenario in which interaction between the 5 leader and a 3 region is required for translation. This is illustrated by the RNA genome of Satellite tobacco necrosis ne- crovirus STNV; [26,27] and of the PAV isolate of Barley yellow dwarf luteovirus BYDV-PAV; [28,29] that have neither cap nor VPg at their 5 end, and neither polyA nor tRNA-like structure at their 3 end. Efficient translation in vitro of STNV RNA which contains a 3 UTR of over 600 nucleotides, requires both the 5 leader and a translational enhancer located within the 3 UTR, just down- stream of the ORF [26,27], and which forms an extended stem-loop structure. Possible base-pair- ing interactions between the 5 leader and the 3 enhancer have been proposed based on comple- mentarity between nucleotides within these re- gions. Removal of the translational enhancer dramatically reduces translation, but translation efficiency can be restored by capping the RNA. Mutations that destroy the translation capacity of the enhancer increase the amount of eIF4F re- quired for translation. In BYDV-PAV, a 3 translational enhancer which is located in the intergenic region between ORFs 5 and 6 the two distal ORFs on the viral genome, is required for translation of the 5-prox- imal ORF in vivo and in vitro [30]. The enhancer decreases the concentration of eIF4F needed for maximum translation of uncapped RNA, and ap- pears to mimic the cap in translation initiation. As in STNV, it is likely that the requirement of a 3 translational enhancer for efficient translation of the 5 ORF of BYDV-PAV is related to the lack of either a cap or a VPg at the 5 end of the genome [29]. No protein has been described that might be required for such translation regulation in the case of STNV or BYDV-PAV. 4 . 1 . 1 . 6 . Viral encoded transacti6ators. These are viral-encoded proteins that specifically enhance translation of a downstream ORF in a bicistronic or a polycistronic mRNA, probably by stimulating reinitiation of translation. Transactivation activity has been demonstrated in the case of several Caulimoviruses; the most thoroughly investigated transactivator is that of CaMV [4]. 4 . 1 . 1 . 7 . Cellular initiation factors. These factors play a crucial role in the efficiency of translation that can be enhanced in viral mRNAs in which one of the cis-elements in the RNA is lacking or mutated. This is for instance the case of the STNV RNA as mentioned above. The influence of these factors will not be discussed further since they have been extensively reviewed recently [31]. 4 . 1 . 2 . Leaky scanning Even more complex is regulation in the case of polycistronic mRNAs, and such mRNAs are ex- tremely frequent among viruses. Leaky scanning is the general mechanism used by viruses in such circumstances. This mechanism is most certainly facilitated if the AUG codon of the 5-proximal ORF is in a less favorable context than the AUG of the second ORF. Here, various genome organi- zations can be found. The two ORFs can be consecutive on the mRNA Fig. 2Aa, and in this case, termination of translation of the 5-proximal ORF is presumably followed by reinitiation at the second ORF. This situation might be facilitated if the 40S ribosomal subunits have remained on the mRNA following Fig. 2. Regulation of gene expression at the level of initiation of translation. A Leaky scanningreinitiation. a Consecu- tive ORFs; the transactivator TAV facilitates reinitiation of the second ORF. b In-frame initiation. c Overlapping ORFs. B Shunting. Curved arrow indicates ‘jumping’ of ribosomes to major ORF. C Internal ribosome entry. Exam- ple of the crTMV sgRNAs. The position of the internal ribosome entry site IRES on the I 2 RNA within the MP ORF of crTMV is indicated. Not to scale. All other indica- tions are as in Fig. 1. encountered strategy among plant viruses. How- ever, it is encountered in the well-studied situation of the MP of Cowpea mosaic comovirus [33]. It is also observed in vivo in the second ORF of RNAb of BSMV. Two proteins are produced from two in-frame AUG codons. By far the most frequent examples of leaky scanning concern overlapping ORFs that are in different reading frames Fig. 2Ac. The overlap- ping regions can in certain cases be extremely long. A large number of RNA containing plant viruses resort to this strategy. A well documented example is that of Peanut clump furovirus RNA2 [34]. Here the AUG codon of the second ORF overlaps the UGA termination codon of the first ORF which codes for the CP. It has been shown that the second ORF is initiated in vitro by con- text-dependent leaky scanning. A similar mecha- nism probably accounts for the synthesis of the two overlapping proteins contained in sgRNA1 of BYDV [35] and Potato leafroll luteovirus [36]: in both cases, the shorter ORF is nested within the longer CP ORF. Triple gene blocks TGB are a group of three proteins whose genes generally overlap on the genome. They are encountered in Potexviruses, Carlaviruses, Furoviruses and Hordeiviruses, and are believed to be important for virus movement through the plant. In BSMV RNAb [37] and in Potato potexvirus X PVX [38], two 3 coterminal sgRNAs are required for the production of the three TGB proteins: the first ORF of the TGB is translated from a functionally monocistronic sgRNA, whereas the second and third ORF are translated from a functionally bi- cistronic sgRNA. In BSMV and in PVX, the initiation codon of the second ORF of the TGB is in a less favorable context for initiation than is that of the third ORF, and it has been demon- strated that leaky scanning is responsible for translation of the third ORF [37]. 4 . 1 . 3 . Shunting In certain cases, a shunt mechanism allows the scanning ribosome to ‘ignore’ certain regions within the leader sequence of an mRNA that can contain short ORFs, jumping, so to speak, to a downstream, longer ORF Fig. 2B. This mecha- nism transfers the ribosome from a donor to an acceptor site on the mRNA, without involvement of mRNA scanning between these two sites. Shunting may be favored by the presence of exten- translation of the upstream ORF. Although not very common among plant viruses, it has been clearly demonstrated in the case of CaMV [4]. Translation of downstream ORFs is facilitated by the action of the viral transactivator, the product of ORF VI. The genome can contain one ORF with two in-frame initiation codons Fig. 2Ab. This leads to the synthesis of two proteins that are identical over the total length of the shorter protein. Trans- lation of the shorter protein is initiated by ribo- somes that by-pass the initiation codon of the longer protein by a context-dependent leaky scan- ning mechanism [32]. This is not a frequently sive secondary structures within the leader se- quence. This mechanism is believed to account for the synthesis of ORF I in the CaMV [39], and in the RTBV [20] transcripts. 4 . 1 . 4 . Internal ribosome entry Interesting cases have recently been reported on the expression of a downstream ORF the CP in the cases described in a dicistronic mRNA. In PVX, a sgRNA contains two non-overlapping ORFs, the 5 proximal ORF coding for an 8-K protein, and the distal ORF coding for the CP. This sgRNA is responsible for the synthesis of both proteins in vitro and in vivo [40]. Although one cannot exclude the possibility that expression of the CP could result from termination of 8-K synthesis followed by reinitiation, the most likely mechanism proposed to account for expression of the CP, is by internal ribosome entry. In a cruci- fer-infecting TMV crTMV, the I 2 sgRNA con- tains the information for the MP and the CP Fig. 2C, the ORF of the latter overlapping the MP ORF [41]. In PVX as in crTMV, the dicistronic mRNA assay as well as other assays has revealed that the region upstream of the CP ORF is pre- sumably responsible for initiation of CP synthesis by an internal ribosomal entry mechanism. Since the studies with crTMV were performed in vitro, they do not exclude the possibility that in vivo a traditional mode of CP expression via the CP mRNA also operates to produce the CP. In agree- ment with this possibility is the demonstration that two sgRNA species corresponding in size to the I 2 and the CP sgRNA are detected in plants infected with crTMV. 4 . 2 . Elongation Viruses also regulate synthesis of their proteins at the level of elongation, and this is achieved by way of frameshifting rev. in Refs. [5,42,43] . This strategy leads to the synthesis of two proteins, a frame and a transframe protein, that are identical in their N-terminal region up to the point of frameshift, but differ in their C-terminal region. The level of synthesis of the frame protein always exceeds that of the transframe protein. Schemati- cally, frameshift appears to result from movement of the ribosome by one nucleotide on the mRNA, either in the 5 or in the 3 direction, leading to a − 1 or a + 1 frameshift event, respectively. A number of signals in the RNA are required for − 1 frameshift. These are 1 a heptanucleotide ‘slippery’ sequence where frameshift occurs, 2 a hairpin structure following the heptanucleotide that in many instances can form a pseudoknot with downstream RNA sequences, and 3 a spacer region of four to nine nucleotides between the heptanucleotide sequence and the hairpin structure. Mutations or removal of any of these elements abolishes frameshift. Cases of − 1 frameshift are frequent among plant RNA viruses, and have been reported for Carlaviruses, Di- anthoviruses, Enamoviruses, Luteoviruses and Sobemoviruses, where they are required for syn- thesis of the RdRp. A + 1 frameshift event requires a slippery run of bases and a rare or ‘hungry’ codon on the ribosomal A site rev. in Ref. [44] . This mecha- nism has been postulated but as yet not demon- strated in the case of the Closteroviruses rev. in Ref. [5]. 4 . 3 . Termination At the level of termination, regulation can occur by readthrough. An in-frame termination codon in an mRNA normally dictates termination of trans- lation. However, ever more examples are being reported in which termination codons are occa- sionally recognized by tRNAs known as suppres- sor tRNAs rev. in Refs. [5,42]. This phenomenon referred to as suppression of termination, repre- sents a means of regulating synthesis of specific proteins, and is frequently encountered in plant viruses. It has been postulated or demonstrated among the Carmo6iridae, Enamoviruses, Furoviruses, Luteoviruses, Tobamoviruses, To- braviruses, Tombusviruses and Necroviruses, and also in an as yet unclassified virus, Oat chlorotic stunt virus. Readthrough produces two proteins, a stopped and a readthrough protein, that are iden- tical over the total length of the stopped protein. As in frameshift, the level of stopped protein produced largely exceeds the level of the readthrough protein. Among plant viruses, readthrough leads to the production of the RdRp or of a CP-fusion protein. In the case of the CP-fusion protein, the readthrough protein is re- covered in the virus particles and is needed either for encapsidation or for transmission of the virus by its insect vector. Until fairly recently, only suppressible UAG and UGA codons had been described. However suppression at the level of a UAA codon has been proposed for Beet soil-borne furovirus [45] and for Beet virus Q, a furo-like virus [46]. In addition to the termination codon, other elements on the mRNA are required in cis for efficient readthrough. In the case of TMV RNA, the na- ture of the two codons following the suppressible UAG codon are crucial for efficient readthrough [47,48]. The requirements in BYDV are totally different. Here, two elements, both located down- stream of the suppressible UAG codon terminat- ing the CP gene are mandatory for readthrough in vitro and in vivo [49]. The proximal element is located six to 15 nucleotides downstream of the UAG, and is composed of 16 repeats of the se- quence CCN NNN N: any nucleotide; deletion of the 5 proximal third of these repeats dramati- cally reduces readthrough. The distal element is about 60 nucleotides long and is located nearly 700 nucleotides downstream of the UAG codon, in the readthrough ORF. Deletions within this element also strongly impair readthrough. The distal element is well conserved among Lute- oviruses and in Pea enation mosaic enamovirus, lending weight to the possibility that a similar role may also be ascribed to the corresponding region in these viruses. It will be interesting to establish whether the proximal and distal regions interact by long-distance base-pairing. As opposed to the situation observed in animal viruses in which a hairpin and even a pseudoknot structure down- stream of the suppressible termination codon are frequently required for readthrough, among plant viruses there has to date been no report that regions downstream of the suppressible termina- tion codon could adopt secondary structures im- portant for efficient readthrough. tRNAs have been isolated from various plant tissues that act as suppressor tRNAs and misread termination codons in trans [5]. These are two Tyr accepting tRNAs for the UAG codon in TMV RNA, as well as a Trp and a Cys accepting tRNA for the UGA codon in the Tobacco rattle to- bravirus RNA. To date, no suppressor tRNA has been isolated that specifically recognizes suppress- ible UAA codons. Nevertheless, the fact that mu- tating the suppressible UAG codon in TMV RNA to a UAA codon leads to virion formation in plants, suggests that a tRNA is present in the host that can recognize UAA or UAG containing TMV RNA [50].

5. Regulation of gene expression at the post-translational level