The 6.3-kb (+)ssRNA genome ofTMV encodes at least four proteins, of which the 5' proximal open reading frame (ORF) encodes a 126 kDa protein terminated by an amber stop codon that when suppressed yields a read-through 183 kDa protein (Goelet et al. 1982; Pelham 1978). Both the 126 kDa and the 183 proteins represent essential replicase components, containing methyl-transferase and helicase domains as well as a polymerase domain on the read-through portion of the 183 kDa ORF (Buck 1999; Koonin and Dolja 1993). The two additional proteins, the 30 kDa movement protein (MP) and the 17.5 kDa capsid protein are dispensable for replication but essential for cell-cell spread of infection and RNA encapsidation, respectively (for review, Heinlein 2002).
For TMV, the virus replication occurs on amorphous proliferations of membranes, previously termed viroplasms or X-bodies, which were initially observed in infected plant cells by Ivanowski (1903). Electron microscopy revealed that these inclusion bodies contained ribosomes, viral RNA, tubules, and 126/183 kDa replication proteins (Esau and Cronshaw 1967; Hills et al. 1987; Martelli and Russo 1977; Saito et al. 1987). Additionally, membrane fractions from TMV-infected cells also contained active replicase complexes capable of synthesizing both plus- and minus-strand viral RNAs in a template-dependent fashion (Osman and Buck 1996).
The use of GFP later revealed that TMV replication complexes localized into large, irregularly shaped, ER-derived structures (especially the cortical ER) containing viral RNA and also the MP (Heinlein et al. 1998; Mas and Beachy 1999; Reichel and Beachy 1998). Mas et al. (1999) further established that the MP was not required for association of the viral RNA with the ER, but was required for the formation of the large irregular bodies. Structural studies recently confirmed the membrane-binding properties of the MP (Brill et al. 2000, 2004). It has been suggested that two highly hydrophobic regions conserved among tobamovirus MPs, the domains I (residues 56-96) and II (residues 125-164) are responsible for the behavior of the MP as an integral membrane protein (Moore et al. 1992; Reichel and Beachy 1998), including its association with cortical, cytoplasmic, and perinuclear ER (Heinlein et al. 1998; Mas and Beachy 1999).
As was the case for the MP, homology searches of the 126 kDa protein failed to identify any potential ER retention motifs or obvious membrane-spanning domains (dos Reis Figueira et al. 2002). However, the TMV 126/183 kDa replicase proteins encode a predicted bipartite nuclear localization signal (NLS) at amino acids 29 to 47 that is conserved within the genus Tobamovirus as well as a potential 21 amino acid amphipathic helix between residues 708-728 (dos Reis Figueira et al. 2002). Interestingly, results from transient expression studies have demonstrated that:
1. The NLS present within the 126/183 kDa protein could function to nuclear localize large polypeptides
2. The NLS domain is necessary for TMV replication
3. The NLS is required but not sufficient for the formation of ER-associated inclusion bodies
4. The full-length 126 kDa protein associates with the ER in the absence of other viral proteins and components (dos Reis Figueira et al. 2002)
Whether the 21 amino acid amphipathic helix is involved in the retention of the 126 kDa protein to the ER is still not known. It has been proposed that a phenylalanine at one end of the helix could function in membrane attachment in a fashion similar to the alphavirus Semliki forest virus nsP1 protein (Lampio et al. 2000). Alternatively, the membrane retention of the 126 kDa could be conferred via a membrane-bound host protein.
In view of the retention of the TMV replication complex by host proteins, Ishikawa and colleagues have identified three Arabidopsis genes, TOM1, TOM2A and TOM3, that are required for efficient tobamovirus replication (Tsujimoto et al. 2003; Yamanaka et al. 2000, 2002). AtTOMl and AtTOM3A share a high degree of similarity (56% identical). They are predicted to be seven-pass transmembrane proteins and are likely to share a parallel and essential function in tobamovirus multiplication (Yamanaka et al. 2002). AtTOM2A is predicted to be a four-pass transmembrane protein. Remarkably, neither of these TOM proteins possesses well-known sorting signals to specific organelles (Tsujimoto et al. 2003; Yamanaka et al. 2000). AtTOM1 and AtTOM3 have been shown to interact with AtTOM2A and with the he-licase domain of the 126/183 kDa proteins (Tsujimoto et al. 2003; Yamanaka et al. 2000), suggesting that TOM proteins are constituents of the replication complex of tobamoviruses and play important roles in the formation of the complex on ER membranes where they colocalize. In support of this hypothesis, subcellular fractionation patterns revealed that the viral RdRp activity coincided well with that of both the membrane-bound 126/183 kDa proteins and TOM1-TOM2A proteins (Hagiwara et al. 2003). However, confocal microscopic analyses of the GFP-tagged TOM proteins showed that TOM1 and TOM2A were predominantly targeted to the vacuolar membranes and not to the ER (Hagiwara et al. 2003). Therefore, whether TMV replication complex is associated with the tonoplast, as strongly suggested by the latter findings, or with the ER still needs to be determined.
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