Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an EPSIN N-TERMINAL HOMOLOGY protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein NEVERSHED/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects. Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection. These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.Higher eukaryotes are involved in a continuing battle against viruses. To minimize the effects of viral infection, a number of defence mechanisms have been developed based on the recognition of specific molecular patterns produced only in infected cells (Plasterk, 2002). This is the case with double-stranded RNA (dsRNA) molecules, which are not normally found in eukaryotic cells but are generated as an intermediate molecule during virus replication (Hutvagner & Zamore, 2002). In mammalian cells, dsRNA appears to play a major role in the induction of the interferon (IFN) response following viral infection (Stark et al., 1998). In recent years, another dsRNA-mediated defence mechanism known as RNA silencing has been described (Dougherty & Parks, 1995;Montgomery & Fire, 1998).RNA silencing is a sequence-specific RNA degradation process that leads to elimination of the targeted RNA mediated by cytoplasmic nucleases and plays a natural antiviral role in plants (for reviews, see Baulcombe, 2002;Carrington et al., 2001;Vance & Vaucheret, 2001;Vazquez Rovere et al., 2002;Waterhouse et al., 2001). Recently, this mechanism of antiviral defence has also been reported in insect cells (Li et al., 2002).In response to these types of host antiviral defences, it is not unexpected that viruses have devised counteracting mechanisms that interfere with them at different levels. Thus, many animal viruses are known to prevent or inhibit IFN-mediated defence (García-Sastre, 2001). The same is true for RNA silencing: many plant viruses (Carrington & Whitham, 1998;Li & Ding, 2001;Voinnet et al., 1999) and one insect virus (Li et al., 2002) have been shown to encode silencing suppressor proteins. Nearly 20 silencing suppressors described so far show no sequence similarity and hence appear to have evolved independently to overcome silencing-mediated defence. Thus, it is not easy to predict which protein is going to behave as an RNA silencing suppressor based on sequence analysis.Since this RNA silencing has been described in species from different kingdoms (fungi, animals and plants), it has been proposed that it can also play an antiviral role in mammalian cells (Cullen, 2002;Gitlin et al., 2002). Here, we have investigated whether the NS1 protein, a multifunctional protein of human influenza A virus, could act as an RN...

SUMMARY Subisolates segregated from an M‐type Plum pox virus (PPV) isolate, PPV‐PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site‐directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV‐PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.