The cucumber mosaic virus (CMV) replication complex has previously been shown to associate with cellular membranes. However, it remains unknown whether any host factors participate in this process. In this study, five groups of Arabidopsis tonoplast intrinsic protein (TIP) genes were isolated and the proteins they encoded were evaluated with regard to their interactions with CMV proteins. TIP1 and TIP2 were found to interact with the CMV 1a protein in the Sos recruitment system, whereas no interactions with the other three TIP subgroups were observed in this assay. The interaction of CMV 1a with the TIP1 and TIP2 proteins was confirmed via co-immunoprecipitation assays. Additionally, CMV 1a co-localized with TIP1 and TIP2 in transfected Arabidopsis protoplasts. The findings of this study suggest that members of two TIP subfamilies might affect CMV replication via interaction with CMV 1a in the tonoplasts.Eukaryotic positive-strand RNA virus replication is known to occur in close association with intracellular membranes. Thus far, no exceptions to this phenomenon have been observed. This clearly indicates the pivotal role of membranes in the formation and operation of viral RNA replication complexes. Direct evidence for the importance of membranes in the process of viral RNA replication has been obtained in several cases (Barton & Flanegan, 1993;Molla et al., 1993;Wu et al., 1992), and different viruses appear to employ different types of cellular membranes (Bienz et al., 1987;Froshauer et al., 1988; Kujala et al., 1999;Pedersen et al., 1999;Schlegel et al., 1996;Suhy et al., 2000;van der Meer et al., 1998).Some viral proteins, including the flock house virus protein A (Miller & Ahlquist, 2002) and hepatitis C virus 4B protein (Hugle et al., 2001) have been identified which harbour transmembrane domains and target replication complexes to intracellular membranes. However, the wide variety of intracellular membrane compartments employed by different positive-strand RNA viruses, as well as the variance inherent in their specific targeting behaviour, indicates that individual viruses may require unique host factors which are associated with specific intracellular membranes (Tereba & Lai, 1982). Despite these expectations, specific information regarding such host factors is currently limited to only a few examples.Cucumber mosaic virus (CMV) replication occurs in association with the vacuolar membrane, also known as the tonoplast (Cillo et al., 2002). Protein 1a, encoded by RNA 1, has a putative methyltransferase domain and helicase domain, and has been recognized as a component of the isolated CMV replicase (Hayes & Buck, 1990). It colocalizes to the tonoplast with protein 2a which harbours an RNA-dependent RNA polymerase motif (Cillo et al., 2002).The tobamovirus multiplication 1 (TOM1) protein and its homologues TOM3 and TOM2A are required for the efficient replication of Tobacco mosaic virus (TMV), but not for the multiplication of either CMV or Turnip crinkle virus in Arabidopsis. They are transmembrane proteins...

Using a yeast two-hybrid system, we identified a plant cellular factor that interacts with the proteins of the Cucumber mosaic virus (CMV). Initially 14 candidate genes were isolated from Nicotiana tabacum, using a full-length CMV 1a gene as bait. Among the candidate genes, two were encoding thaumatin-like proteins (TLP), and were designated as Nicotiana tabacum thaumatin-like protein 1 (NtTLP1). Consistent with this observation, recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein and coat protein, in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation.