Morphogenesis Of Replication Factories And Host Responses In Beet Black Scorch Virus-infected Nicotiana Benthamiana

Most plant viruses belong to positive-sense RNA virus and establishment of virus replication complexes (VRCs) of all well-studied positive-strand RNA viruses depends on the various host endomembranes. Despite that VRCs of several viruses have been well-characterized, the underlying mechanism and machinery for VRCs formation are still elusive. Beet black scorch virus (BBSV), a necrovirus in the Tombusviridae, is a positive single-strand RNA virus. In this work, the location, formation and three dimensional (3D) architecture of BBSV-induced replication factories in Nicotiana benthamiana was studied. Meanwhile, the involvement of unfolded protein response (UPR) and host factors during BBSV infection were also explored.Endoplasmic reticulum (ER) aggregation was observed in BBSV-infected N. benthamiana cells using confocal laser scanning microscopy (CLSM), and transmission electron microscopy (TEM) analysis showed that BBSV infection leads to the convolution and aggregation of ER. Intriguingly, many spherules formed by ER membrane invagination were frequently observed in BBSV-infected cells. Bimolecular fluorescence complementation (BiFC) assays also indicated that p23/p82complexes colocalized well with the ER aggregates. Moreover, Immunogold electron microscopy showed that the auxiliary replication protein p23was specifically localized on the ER and ER-associated spherules, and replication intermediate dsRNA localized specifically in the ER-associated membrane spherules. All the results above revealed that the ER-associated membrane vesicles are the sites for VRCs assembly and BBSV replication.Transient expression of the auxiliary replication protein p23in N. benthamiana cells was always accompanied by ER aggregation in CLSM assays. TEM analysis also showed that expression of p23led to ER convolution and aggregation. Conversely, there were no obvious changes of the ER membrane in cells expressed BBSV other proteins. These results indicated that expression of p23alone could alter the membrane structure of ER, although it was not sufficient to form spherules seen during BBSV infection. Membrane flotation assays and biochemical analysis revealed that p23is a transmembrane protein, and mutational analysis showed that the transmembrane domains locate to the N terminal of p23, which is essential for its ER location and subsequent remodeling of the ER.The3D architecture of BBSV-induced replication factories was reconstructed using electron tomography, which, to our knowledge, was the first report in virus-infected plant cells. Comparing the3D architecture of BBSV VRCs with previously reported ones in animal cells showed that it has some distinct features besides the common characteristics presented by other VRCs. The interior of virus replication vesicle connected with the surrounding cytoplasm through a5-7nm neck-like channel. In addition, vesicle packets were often connected to each other via tubes, and the shape of the fibrillar materials in the spherules appeared in a diverse state. The3D ultrastructure would aid for better understanding the mechanisms underlying the replication of BBSV as well as other positive-strand RNA viruses. Overexpression of p23whether by Potato virus X-based vector or CaMV35S promoter could induce cell death in N. benthamiana leaves. And ectopic expression of BiP could compromise p23-induced cell death, suggesting that p23-induced cell death was related with unfolded protein response (UPR). The expression of six UPR-related genes(bZIP60, BiP, PDI, CRT, CAM and SKP1) at different time points during BBSV infection was examined in northern blot assays and results indicated that different UPR-related genes were regulated in a time-dependent manner. Down-regulaton of bZIP60in N. benthamiana significantly decreased BBSV accumulation and the amount of ER aggregates, suggesting that UPR was necessary for BBSV infection. This is the first report of a plant virus auxiliary replication protein that was able to induce UPR during viral infection.Finally, host factors that involved in BBSV replication were investigated. Preliminary results showed that Hsp70and Hsp90could be up-regulated in BBSV-infected N. benthamiana, and down-regulation of Hsp70and Hsp90in N. benthamiana significantly impaired BBSV replication. Both Hsp70and Hsp90could interact with p23directly as evidenced by BiFC and GST pull-down assays. These results suggested that Hsp70and Hsp90were important host factors involved in BBSV replication. More experiments should be performed to elucidate how these two factors exert functions during BBSV infection.