Cowpea Mosaic Virus

CPMV RNA1 and RNA2 express large polyproteins, which are proteolytically cleaved into different proteins by the RNAl-encoded viral 24-kDa proteinase (24 K). The proteins encoded by RNA1 are necessary and sufficient for virus replication. The function of RNAl-encoded proteins functions has been attributed as follows. The 32-kDa protein (32 K) is a hydrophobic protein which does not contain a motif common to other positive-strand RNA viruses outside the Comoviridae. It is involved in the regulation of processing of RNAl-encoded polyprotein and is required as a cofactor in the cleavage of the RNA2 polyprotein (Peters et al. 1992). The 58 K is able to bind ATP via a conserved nucleotide-binding motif and has been proposed to be a viral helicase (Peters et al. 1994). The 28 amino acid VPg is linked to the 5' end of the RNA via a serine residue and is likely involved in initiation of viral RNA synthesis (Carette et al. 2001). The 24 K protease is structurally similar to the trypsin-like family of serine proteases with residues His40, Glu76, and Cys166 representing the catalytic triad of the active site (Dessens and Lomonossoff 1991). The 87 K has a domain specific to RNA-dependent RNA polymerases; however, the 110 K (87 K plus 24 K) is the only viral protein present in highly purified "replication complexes" capable of elongating nascent viral RNA chains, suggesting that fusion to 24 K is required for replicase activity (Eggen et al. 1988).

Upon infection of cowpea plants with CPMV, a typical cytopathic structure is formed, often adjacent to the nucleus and consisting of an amorphous matrix of electron-dense material that is traversed by rays of small membranous vesicles (De Zoeten et al. 1974). Both subcellular fractionation experiments of CPMV-infected leaves and autoradiography, in conjunction with electron microscopy, on sections of CPMV-infected leaves treated with [3H] uridine revealed that the membranous vesicles are closely associated with CPMV RNA replication (De Zoeten et al. 1974; Eggen et al. 1988; Zabel et al. 1974). Curiously, more detailed analysis later showed that the bulk of the replication proteins in CPMV-infected cells immunolocalized not to the vesicles, but to the adjacent electron-dense structures, suggesting that only a small part of the RNA1-encoded proteins contributes to the active replication complexes (Wellink et al. 1988). The accumulation of replication proteins into the electron structure is likely to depend on interactions between the 32 K and the 60 K precursor (VPg + 58 K) since deletion of the 32 K coding region, single amino acid substitution within the VPg, or the NTP binding of the 58 K protein all abolished formation of the dense bodies (Carette et al. 2001; Peters et al. 1994; van Bokhoven et al. 1993). The significance of these electron-dense structures for viral replication remains unclear as a mutation in the VPg was identified that prevented the formation of the cytopathic structures, without abolishing viral replication (Carette et al. 2001). They may simply represent deposition of inactive nonstructural protein or sites where overproduced proteins accumulate (Carette et al. 2001).

In an attempt to identify the membrane that contributes to CPMV replication, it was demonstrated through the use of transgenic Nicotiana benthami-ana plants expressing ER- or Golgi-targeted GFP that CPMV infection causes a strong proliferation of ER membranes without affecting the structure of the Golgi stacks (Carette et al. 2000). ER modifications start at the cortical ER network and culminate with the apparition of a large region of densely packed ER membranes, often near the nucleus (Carette et al. 2000). Treatment with cytoskeleton inhibitor further revealed that intracellular trafficking of replication complexes to the large juxtanuclear structure occurs via association with the actin cytoskeleton and not the microtubular network (Carette et al. 2002a). The combined use of fluorescent in situ hybridization (FISH) and ER-GFP marker also showed that during the course of an infection, CPMV RNA colocalizes with the 110 K viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, supporting the view that ER membranes act as a source for the small membranous vesicles (Carette et al. 2002a). Not surprisingly, changes in ER morphology could be attributed to RNA1 alone. Thus, expression of RNA1-encoded proteins in insect cells, by using a baculovirus expression system, showed that 60 K, but not 110 K, is able to induce and associate with small membranous vesicles in the cytoplasm in these cells (van Bokhoven et al. 1992). More significantly, expression of individual viral proteins in N. benthamiana epi dermal cells using a viral vector revealed that both 32 K and 60 K, when fused to GFP, associate to membranes and induce rearrangement of the ER (Carette et al. 2002b). The alterations of the ER morphology resembled the proliferations that occur in CPMV-infected cells, although some differences could be observed. In particular, the GFP-32 K was present mainly in the cortical ER, whereas GFP-60 K was found mainly in the nuclear envelope, the plastidial membrane, and aggregates presumably derived from the ER. Other RNA1-encoded proteins, the 110 K polymerase and the N-terminal cleavage product 24 K proteinase, behaved as freely soluble proteins when expressed in isolation (Carette et al. 2002b). The localization signals that target the 32 K and 60 K, and probably also the replication complexes to ER membranes remain to be identified. It has been suggested that the three stretches of hydrophobic amino acids at the C-terminal end of the 32 K contribute to the membrane attachment (Carette et al. 2002b). Similarly, the 60 K contains two hydropho-bic domains that are conserved among the comoviruses: an amphipathic helix at the N terminus (amino acids 45 to 61) and a 22-amino-acid stretch of hy-drophobic residues (amino acids 544 to 565) at the C terminus immediately upstream of VPg (Carette et al. 2002b; Peters et al. 1992).

How ER membranes contribute to the formation the CPMV-induced vesicles is still a matter of debate. Immunogold labelling experiments on CPMV-infected tobacco leaf cells showed that they contained a relatively low amount of the luminal GFP-ER marker protein, indicating that luminal ER proteins are excluded (Carette et al. 2000) during generation of these vesicles. Additionally, it seems that the vesicle formation in CPMV-infected cells involves de novo membrane synthesis rather than a modification of preexisting membranes, as it was found that cerulenin, a fungal antibiotic which prevents de novo phospholipid biosynthesis and exerts its action by specifically inhibiting document ^-ketoacyl-acyl carrier protein synthase (Moche et al. 1999), proved to be a strong inhibitor of CPMV RNA replication (Carette et al. 2000). Carette et al. (2002a) suggested that the vesicles may form as a result of the unfolded-protein response (UPR). This response can occur after various biochemical and physiological stimuli that affect ER homeostasis and impose stress to the ER, subsequently leading to accumulation of unfolded or misfolded proteins in the ER lumen (Shen et al. 2004). However, attempts to demonstrate the up-regulation of BiP that serves as a hallmark in the UPR remained unsuccessful (Carette et al. 2002a). Interestingly, a yeast two-hybrid search of an Arabidopsis cDNA library showed that the C-terminal domain of 60 K protein interacts with two vesicle-associated proteins (termed VAP27-1 and VAP27-2) that belong to the VAP33 family of SNARE-like proteins (Carette et al. 2002c). These host proteins also localized with the 60 K in CPMV-infected protoplasts. Carette et al. (2002c) suggested that VAP27-1 and VAP27-2 could play an important role in vesicular transport to or from the ER and may also act as a membrane anchor for the virus replication complex. Alternatively, interference of 60 K with VAP function could contribute to the ER vesiculation process seen during infection. However, experimental evidence demonstrating the involvement of VAP27-1 and VAP27-2 in CPMV replication is still missing.

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