Degradation of Tobacco Mosaic Virus Movement Protein by the 26S Proteasome

Abstract

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER).During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and clasto-lactacystin-␤-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.To spread infection from cell to cell, plant viruses must cross the rigid cell wall between cells by exploiting cytoplasmic bridges referred to as plasmodesmata. These cytoplasmic channels maintain continuity of the plasma membrane, the cytoplasm, and the endoplasmic reticulum (ER) between neighboring cells (30,33,43). Plasmodesmata enable neighboring plant cells to exchange small signal molecules and metabolites but under normal conditions exclude molecules of more than 1 kDa. Thus, plasmodesmata must be modified in order to allow the spread of infection from cell to cell. During replication, plant viruses produce proteins that facilitate cell-cell transport, referred to as movement proteins (MP). MPs have been demonstrated to directly or indirectly modify the size exclusion limit of plasmodesmata to permit viral spread (10,30,56).Tobacco mosaic tobamovirus (TMV), a plus-sense RNA virus, encodes four proteins. The 126-kDa replicase and a 183-kDa replicase readthrough product are involved in genome amplification and are translated from genomic RNA, while the 17.5-kDa coat protein (CP) and the 30-kDa MP are translated from subgenomic RNAs.Although there have been very detailed investigations of the function of the TMV MP and its intracellular localization, few studies have dealt with the regulation of virus spread and the involvement of the MP in this process. Genetic studies involving transgenic plants expressing functional and nonfunctional MP variants, and studies involving viruses that contain mutations in the MP, established the role of the MP in facilitating cell-cell spread of infection (5,9,10,14,20,32). Electron microscopy, fluorescence microscopy, and cell fractionation were used to elucidate the intracellular localization of MP and MP-GFP fusion proteins (1,18,19,31,38,45,46). These studies established that the MP is tightly associated with plasmodesmata in transgenic plants and that during virus infection there is a transient association with several compartmen...

MPB2C, a Microtubule-Associated Plant Protein Binds to and Interferes with Cell-to-Cell Transport of Tobacco Mosaic Virus Movement Protein

“…This pathway is not involved in MP30 cell-to-cell transport but might be assigned for degradation as previously suggested (Padgett et al, 1996;Mas and Beachy, 1999;Reichel and Beachy, 2000;Gillespie et al, 2002). When MPB2C is present at endogenous levels, a number of MP30 molecules produced in infected cells escape the MPB2C involving microtubular accumulation pathway and engage in cell-tocell transport.…”

“…On the other hand, microtubules have also been implicated in MP30 degradation (Padgett et al, 1996;Mas and Beachy, 1999;Reichel and Beachy, 2000). In line, an MP30 mutant that escapes accumulation at microtubules but displays enhanced cell-to-cell movement was recently described (MP R3 ; Gillespie et al, 2002), indicating that accumulation at microtubules is detrimental for MP30 movement.…”

“…In line with this model, microtubules have been functionally implicated in guiding vRNA-MP30 complexes toward plasmodesmata (Mas and Beachy, 2000; Boyko et al, 2000a Boyko et al, , 2000b. However, an alternate, non-movement-promoting role of microtubules in providing a route toward an MP30 degradation site was suggested (Padgett et al, 1996;Mas and Beachy, 1999;Reichel and Beachy, 2000). This model is supported by the observation that TMV was able to spread in tissues with functionally impaired microtubules and by studies performed with a mutant variant of MP30, MP R3 with strongly decreased affinity to microtubules but increased cell-to-cell movement capacity (Gillespie et al, 2002).…”

Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein

“…Deletions of three amino acids at regular intervals throughout the MP sequence allowed the identification of important functional domains that are involved in the subcellular accumulation of the MP (13). Previous studies also have shown that a polyubiquitinated TMV MP is degraded by the 26S proteasome (14).…”

Binding of monoclonal antibodies to the movement protein (MP) of Tobacco mosaic virus: influence of subcellular MP localization and phosphorylation

“…Within the infected cell, MP is synthesized at the leading edge and degraded at the trailing edge of the infection site (Padgett et al, 1996). Polyubiquitination of MP and subsequent degradation by the 26S proteasome pathway occurs after the early stages of infection (Reichel & Beachy, 2000). However, the mechanisms responsible for the regulation of transient MP expression are not clear.…”