Constitution And Mechanism Analysis Of Membrane Associated Inclusion Bodies Formed In Wheat Yellow Mosaic Virus Infected Plant Cell

Viruses often modify host endoplasmic reticulum or other endomembranes to facilitate biological processes in their life cycle. It is known that infection by viruses belonging to the genus Bymovirus (family Potyviridae) can induce the formation of large membranous inclusion bodies in host cells, but the mechanism by which they are formed and their biological roles in virus genome replication and particle assembly are still unclear.Immuno-electron microscopy of cells infected with the Bymovirus wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein that constituent the major components of membranous inclusion bodies, while NIa-Pro (nuclear inclusion a-proteinase) and VPg (viral protein genome-linked) are likely minor ones. HDEL is an endoplasmic reticulum resident protein identification signal, which can also be immuno-labeled on membranous inclusions. Subcellular localization analyze suggested that PI and P2 associated with the endoplasmic reticulum (ER), while P3 targeted both the ER and the Golgi apparatus. Bioinformatics analyses and chemical experiments showed that P2 is an integral membrane protein and that it depends on the active secretory pathway to form aggregates of ER membranes. Our results also indicated that PI is a peripheral protein, whereas P3 is an integral membrane protein and the transport of P3 from ER to Golgi involves the active secretory pathway. Bimolecular fluorescence complementation (BiFC) assays indicated that P1 interacted with itself and with the most of WYMV encoded proteins except 14K, while P1, P2 and P3 interacted with themselves and between each other. Furthermore, we also analyzed the subcellular localization of VPg and NIa-Pro. VPg-eGFP exclusively localized in the nucleus. NIa-Pro-eGFP had no specific subcellular localization, but diffusely distributed throughout both the cytoplasm and nucleoplasm.Of the WYMV-encoded proteins associated with membranous inclusions, only P2 was able to rearrange ER and form large aggregate structures. Planta and in vitro assays demonstrated that P2 interacted with P1, P3, NIa-Pro or VPg and recruited those proteins into the aggregates. In vivo double immuno-fluorescence labeling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurred in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house genome replication of WYMV.