SYNTHESIS OF VIRAL PROTEINS: TRANSLATION

Plant viruses have evolved several unconventional translational strategies that allow efficient expression of more than one protein from their compact, multifunctional RNAs, as well as regulation of polycistronic translation in the infected plant cell. Here, we review recent advances in our understanding of these unconventional mechanisms, which include leaky scanning, ribosome shunting, internal initiation, reinitiation, stop codon suppression and frameshifting, and compare their characteristics with related phenomena in other systems.

The initiation of translation requires many proteins. It is a process with many stages: initiation, elongation and termination. The initiation is a very complex process.

The initiation step can be of two types:

§  Cap-dependent initiation

§  Cap-independent initiation

Cap-dependent initiation

Cap-dependent initiation is same as in case of eukaryotes. It requires m7GpppNcap structure (where N is any nucleotide) at the 5′-end, a not-very-long unstructured sequence preceding the translation start codon (5′-leader), and a poly(A) tail at the 3′-terminus. These structural features are required for recruitment of the protein synthesis machinery during general translation initiation via the cap-dependent pathway, where the translation start site is chosen by strictly linear scanning of the 40S ribosomal subunit along the 5′-leader starting from the capped 5′-end. This cap and linear ribosome scanning-dependent. Mode of initiation is the main translation initiation pathway in eukaryotes, involving numerous initiation factors (eIFs) and the interplay of a succession of protein-protein and protein-RNA complexes (Hershey and Merrick, 2000).

Step 1. Separation of 80S ribosomes into 40S and 60S ribosomal subunits. The pool of small ribosomal subunits isthen activated by binding of eIF1A, eIF1 and the largest eIF,eIF3 (Peterson et al. 1979; Phan et al. 1998; Chaudhuri et al. 1999; Majumdar et al. 2003). Importantly, eIF3 can support dissociation of 80S in the presence of mRNA or the ternary complex (TC, Met-tRNAiMet/eIF2/GTP) and eIF1 in mammals (Unbehaun et al. 2004; Kolupaeva et al. 2005).

Step 2. Binding of TC to 40S subunit. The 40S ribosomal subunit, together with eIF3, eIF1, eIF1A, eIF5 and the TC, forms a 43S pre-initiation complex. Although eIF3, eIF1 and eIF1A can directly bind 40S, thereby stimulating the formation of the 43S complex, in yeast TC is associated with eIF3, eIF1, and eIF5 in a pre-existing multifactor complex that can interact with the 40S (Asano et al. 2000). eIF2 interacts with eIF3 directly via the eIF3a subunit and indirectly

via eIF5 bridging the two factors.

Step 3. Priming of the mRNA 5′-end cap structure by eIF4F, eIF4A and eIF4B. eIF4F is comprised of the cap-binding factor eIF4E, the ATP-dependent RNA helicaseeIF4A and a scaffold protein eIF4G, which contains binding domains for eIF4E, eIF4A and poly(A)-binding protein(PABP; Sachs 2000; Gross et al. 2003). eIF4A, the DEAD box helicase, participates in ATP-dependent unwinding of the mRNA secondary structure; its RNA melting activity is stimulated by eIF4G and eIF4B (Rogers et al.2002). eIF4G can recruit other factors, including eIF3 and PABP through direct protein–protein interactions. It is thought that eIF4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A in mammals (Jaramillo et al. 1990) and plants (Metz et al. 1999) and mediates binding of mRNA to ribosomes eIF4B can physically interact with eIF3 in yeast and plants (via eIF3g; Vornlocher et al. 1999; Park etal. 2004) and in mammals (via eIF3a; Méthot et al. 1996).PABP binds to the poly(A) tail present at the 3′-end of most cellular mRNAs, and the interaction between PABP and eIF4G brings both termini of an mRNA into close spatial proximity, effectively resulting in mRNA circularization (Wells et al. 1998a).

Step 4. Binding of mRNA to the 43S complex.eIF4G and, apparently, eIF4B potentially serve as organizing centres for loading of the 43S preinitiation complex onto the 5′-end of the mRNA, mainly via interactions between PABP, eIF4G, eIF4B, eIF3, eIF2 and mRNA (Gingras et al. 1999).

Step 5. Scanning of the mRNA leader and start codon recognition. The 43S complex loaded at the capped 5′-endof the mRNA scans the downstream leader sequence until it encounters the first start codon in an optimal initiation context [(A/G)CCAUG(G); Kozak 1987a, 1991]. The scanningprocess of the 43S preinitiation complex requires ATP hydrolysis and is dependent on two eIFs, eIF1 and eIF1A, which are required for the ribosomal complex to locate the initiation codon (Pestova et al. 1998). Start site selectionthen requires cooperation between the scanning ribosome and eIF1, eIF2 and eIF5, which form the 48S preinitiation complex at the optimal start codon. As a result, Met-tRNAiMet will be located at the ribosomal P-site (peptidyl-tRNA binding site on the ribosome), where the anticodon of MettRNAiMet and AUG codon are base paired.

Step 6. 60S subunit joining. As soon as the 48S complex is formed, eIF5 – a GTPase-activating protein – stimulates hydrolysis of eIF2-bound GTP, and eIF2-bound GDP is released from the 48S preinitiation complex (Merrick, 1992). Joining of the 60S subunit also requires an additional factor, termed eIF5B, which has a ribosome-dependent GTPase activity (Pestova et al. 2000). eIF5B catalyses ribosomal subunit joining, and all other translation initiation factors are supposedly released (Unbehaun et al. 2004). The resulting 80S complex is ready to enter the elongation phase of translation. Recycling of eIF2-bound GDP to eIF2-bound GTP is stimulated by eIF2B. The translational machinery of plants, despite having some unique plant-specific factors, closely resembles that of mammals. Although most eIFs are generally similar in all eukaryotes, there are a few striking differences between mammalian and plant translation initiation factors (Browning, 2004). For example, higher plants possess an isozyme form of eIF4F, termed eIF(iso)4F, containing eIF(iso)4E and eIF(iso)4G, which shows preferences for initiation at unstructured non-coding regions (Gallie and Browning, 2001). In the case of eIF4B, there is essentially no conservation at the primary amino acid sequence level between yeast, mammals and plants (Metz et al. 1999). The plant eIF4B contains three RNA binding domains, two binding domains for PABP and eIF4A, and one binding site for eIF(iso)4G (the plant isoform of eIF4G) (Cheng and Gallie 2006). Some conservation between plant and mammalian factors, in regions required for the recruitment of eIF4A and PABP have, however, been suggested (Cheng and Gallie 2006).

Translation elongation

The working elongation cycle of the eukaryotic ribosome is basically similar to that of prokaryotes and consists of three main steps: codon-dependent binding of aminoacyl-tRNA (step 1), transpeptidation (step 2), and translocation (step 3; for a detailed description, see Merrick and Nyborg 2000). The binding sites of aminoacyl-tRNA and peptidyl-tRNA on the ribosome have been designated as the A and P sites, respectively.

Step 1. Binding of the aminoacyl-tRNA to the A-site. At this point the peptidyl-tRNA occupies the P site. The aminoacyl- tRNA, complexed with eEF1 and GTP, enters the ribosome and binds to the mRNA codon located in the A-site of the 80S ribosome. This binding is accompanied by the hydrolysis of a GTP molecule and the release of the eEF1/GDP complex. eEF1 consists of the eEF1A subunit, which binds GTP and elongator tRNA, and eIF1B, a three-subunit complex that is a guanine nucleotide exchange factor for eEF1A. The eEF1 holofactor containing all four subunits is known as eEF1H.

Step 2. Transpeptidation is catalyzed by the ribosome itself and occurs between the aminoacyl-tRNA in the A-site and the peptidyl-tRNA in the P-site, with the peptide C-terminus being transferred to the aminoacyl-tRNA. As a result, the elongated peptidyl-tRNA now occupies the A site while the deacylated tRNA formed in the reaction is relocated to the P site.

Step 3. Translocation. The ribosome interacts with eEF2, a single subunit protein, and GTP, and this catalyzes the displacement of the peptidyl-tRNA (its tRNA residue) along with the template codon from the A site to the P site, as well as the release of the deacylated tRNA from the P site.

During these events, GTP undergoes hydrolysis and eEF2/ GDP is released from the ribosome. At the end of each cycle the peptidyl-tRNA is located in the P site while the next template codon is located in the A site; thus the A site is ready to accept the next aminoacyl-tRNA molecule.

Translation of the mRNA and corresponding polypeptide elongation on the ribosome are achieved by repetition of this cycle.

Translation termination

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes all three termination codons, UAA, UAG, and UGA, at the ribosomal A-site and induces hydrolysis of peptidyl tRNA at the P site (Frolova et al. 1994). As a result, the polypeptide is released from the ribosome. The function of the second termination factor – eRF3 – is not well understood, although it is known to interact with GTP and show GTPase activity in

the presence of ribosomes. There is evidence that eRF3 together with GTP can form a complex with eRF1. Thus, it is the complex eRF1/eRF3/GTP that may be the functional 3 unit required for termination on the eukaryotic ribosome in a GTP-dependent manner (Figure 4).