Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome.

Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.

Caterpillar saliva interferes with induced Arabidopsis thaliana defence responses via the systemic acquired resistance pathway.

Arabidopsis thaliana (L.) Heynh. genotypes limited in their ability to mount either octadecanoid-dependent induced resistance (IR(-)) or systemic acquired resistance (SAR(-)) were used to characterize the roles of these pathways in plant-herbivore interactions. Molecular and biochemical markers of IR were analysed in plants subject to herbivory by caterpillars of the beet armyworm, Spodoptera exigua Hübner, which had either intact or impaired salivary secretions since salivary enzymes, such as glucose oxidase, have been implicated in the ability of caterpillars to circumvent induced plant defences. Transcript expression of genes encoding laccase-like multicopper oxidase [AtLMCO4 (polyphenol oxidase)] and defensin (AtPDF1.2) showed salivary-specific patterns which were disrupted in the SAR(-) mutant plants. The activity of octadecanoid-associated anti-nutritive proteins, such as LMCO and trypsin inhibitor, showed similar patterns. Gene and protein changes parallel plant hormone levels where elevated jasmonic acid was observed in wild-type plants fed upon by caterpillars with impaired salivary secretions compared with plants subject to herbivory by normal caterpillars. This salivary-specific difference in jasmonic acid levels was alleviated in SAR(-) mutants. These results support the model that caterpillar saliva interferes with jasmonate-dependent plant defences by activating the SAR pathway.

Eukaryotic elongation factor 1A interacts with Turnip mosaic virus RNA-dependent RNA polymerase and VPg-Pro in virus-induced vesicles.

Eukaryotic elongation factor 1-alpha (eEF1A) was identified as an interactor of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp) and VPg-protease (VPg-Pro) using tandem affinity purification and/or in vitro assays. Subcellular fractionation experiments revealed that the level of eEF1A substantially increased in membrane fractions upon TuMV infection. Replication of TuMV occurs in cytoplasmic membrane vesicles, which are induced by 6K-VPg-Pro. Confocal microscopy indicated that eEF1A was included in these vesicles. To confirm that eEF1A was found in replication vesicles, we constructed an infectious recombinant TuMV that contains an additional copy of the 6K protein fused to the green fluorescent protein (GFP). In cells infected with this recombinant TuMV, fluorescence emitted by 6KGFP was associated with cytoplasmic membrane vesicles that contained VPg-Pro, the eukaryotic initiation factor (iso) 4E, the poly(A)-binding protein, the heat shock cognate 70-3 protein, and eEF1A. These results suggest that TuMV-induced membrane vesicles host at least three plant translation factors in addition to the viral replication proteins.

Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles.

Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.

The VPgPro protein of Turnip mosaic virus: in vitro inhibition of translation from a ribonuclease activity.

A role for viral encoded genome-linked (VPg) proteins in translation has often been suggested because of their covalent attachment to the 5' end of the viral RNA, reminiscent of the cap structure normally present on most eukaryotic mRNAs. We tested the effect of Turnip mosaic virus (TuMV) VPgPro on translation of reporter RNAs in in vitro translation systems. The presence of VPgPro in either wheat germ extract or rabbit reticulocyte lysate systems lead to inhibition of translation. The inhibition did not appear to be mediated by the interaction of VPg with the eIF(iso)4E translation initiation factor since a VPg mutant that does not interact with eIF(iso)4E still inhibited translation. Monitoring the fate of RNAs revealed that they were degraded as a result of addition of TuMV VPgPro or of Norwalk virus (NV) VPg protein. The RNA degradation was not the result of translation being arrested and was heat labile and partially EDTA sensitive. The capacity of TuMV VPgPro and of (NV) VPg to degrade RNA suggests that these proteins have a ribonucleolytic activity which may contribute to the host RNA translation shutoff associated with many virus infections.