Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with 3'-untranslated region (3'-UTR) of TMV RNA but also directly with RdRp without mediation by the 3'-UTR. The methyltransferase domain of TMV RdRp was indicated to be responsible for the interaction with eEF1A in vitro and in yeast. These results suggest that eEF1A is a component of the virus replication complex of TMV.

The movement protein (MP) of tobacco mosaic virus (TMV) mediates the transport of viral RNA from infected cells to neighboring uninfected cells via plasmodesmata by interacting with putative host factors. To find such host factors, we screened tobacco proteins using the yeast two-hybrid system. NtMPIP1, a novel subset of DnaJ-like proteins, was identified from a tobacco cDNA library, and its specific interaction with TMV MP was confirmed with an in vitro filter-binding assay. In a deletion analysis, using a series of truncated TMV MPs and NtMPIP1s, at least two regions of TMV MP, amino acid residues 65-86 and 120-185, conferred the ability to interact with the C-terminal domain of NtMPIP1, which is thought to be involved in substrate binding. Virus-induced gene silencing of NtMPIP1 significantly inhibited the spread of TMV. Therefore, it is reasonable to consider that endogenous NtMPIP1 is a host factor involved in virus cell-to-cell spread by interacting with TMV MP.

Eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with both the viral RNA-dependent RNA polymerase and the 3'-terminal genomic RNA of tobacco mosaic virus (TMV). In this study, we demonstrated that the down-regulation of eEF1A mRNA levels by virus-induced gene silencing using potato virus X vector dramatically reduced the accumulation of TMV RNA and the spread of TMV infection. The translation activity of the eEF1A-silenced Nicotiana benthamiana leaves was not severely affected. Collectively, these results suggest an essential role of eEF1A in TMV infection.

No abstract

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.