Functional Characterization Of Potyvirus-Encoded Membrane-Associated Proteins

Potyvirus is the largest plant virus family, includes more than 200 species,≈30% of known plant viruses, can be responsible for severe economic losses to agriculture world-wide. Potyvirus contains a single-stranded, positivesense [ss(+)] RNA genome of about 10 kb nucleotides. There is a VPg (viral protein genome-linked) covalently linked to its 5'end and a poly(A) tail at the 3'end. The viral genome contains a single open reading frame (ORF), encoding a large polyprotein of about 360 kDa that is ultimately cleaved into 11 viral protein products. The function of viral proteins are complex. Potyvirus replication mechanism is still not clear. As (+)RNA viruses replicate in association with cellular membranes, it is the foundation for replication to investigate the function of potyvirus-encoded membrane-associated proteins. In this paper, to better understand the replication mechanisms of potyviruses, the functional characterization of potyvirus-encoded membrane-associated proteins (6K2 protein, P3 protein, P3-PiPo protein) were investigated. The main contents were as follows:1,6K2 protein contains a central hydrophobic domain, which has been demonstrated to associate with potyviruses replication mechanisms. We used yeast two-hybrid systerm to screen the SMV host proteins from the model plant Nicotiana benthamiana cDNA library that interact with soybean mosaic virus london strain (SMV-L) encoded proteins, a 6-kDa potyvirus membrane protein (6K2). Yeast two-hybrid screening has identified a total of 22 host candidates that interacted with 6K2. Localization and co-localization with 6K2 of 8 selected host proteins have been observed by confocal microscope. ATO protein was co-localized with 6K2 on the chloroplasts, confirmed by the bimolecular fluorescence complementation assay (BiFC) in planta. So we suggest hypothetical model for that ER-derived membranous vesicles induced by 6K2 protein was trafficed to the chloroplast through interaction with ATO, then the VRC was induced into chloroplast.2,Besides of 6K protein, P3 protein of potyviruses also contained two hydrophobic domains, to be suggested viral membrance protein. To explore the possible function of P3, we investigated the subcellular localization of the Tobacco etch virus (TEV) P3 protein in Nicotiana benthamiana leaf epidermal cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway(COPI and COPII). The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments. Taken together, the possible dual roles of P3 in viral replication and movement suggested that this protein may act as bridge to anchor the membrane-bound VRC close to the actin microfilament.3,Recently, P3-PiPo was discovered, which is an overlap protein produced via ribosomal frameshifting or transcriptional slippage at a highly conserved within the P3 cistron. In order to investigate the possible role of P3-PiPo, we investigated the soybean mosaic virus london strain (SMV-L) viral proteins interactions with P3-PiPo by yeast two-hybrid system. The interaction between SMV-L P3-PiPo and its NIb was detected and supported with Bimolecular fluorescence complementation (BiFC). We further find out the PIPO domain responsible for this interaction by YTHS. The subcellular localization of P3-PiPo protein in Nicotiana benthamiana leaf epidermal cells shown a little punctate inclusions along the plasma membrance. Its co-localization with NIb were detected in karyotheca. When co-expressing P3-PiPo with NIb,6K2-NIa, we can observed P3-PiPo together with 6K2-NIa, accumulating in the viral-derived vesicles beside the choropalst, where is the site of VCR. So we can suggest that P3-PiPo was involved in the virus replication.Taken together, the functional characterization of potyvirus-encoded membrane-associated proteins promoted the research of potyviruses replication mechanisms. These data contain that the hypothetical model for that ER-derived membranous vesicles induced by 6K2 protein playing a key role in potyviruses replication through interaction with ATO, that the possible dual roles of P3 in viral replication and movement, that the P3-PiPo involved in the VCR.