RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.
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This work was supported by the National Science Foundation, MCB-1517751, awarded to PDN.
Nawaz-ul-Rehman, Muhammad Shah; Prasanth, K. Reddisiva; Xu, Kai; Sasvari, Zsuzsanna; Kovalev, Nikolay; Fernández de Castro Martín, Isabel; Barajas, Daniel; Risco, Cristina; and Nagy, Peter D., "Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly" (2016). Plant Pathology Faculty Publications. 59.
S1 Materials and Methods. Supplementary Materials and Methods.
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S1 Fig. Testing Cof1p mutants for their ability to inhibit TBSV repRNA accumulation in yeast.
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S2 Fig. Over-expression of the native Cof1p inhibits TBSV repRNA accumulation in yeast.
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S3 Fig. Confocal laser microscopic images of TBSV-infected plant cells expressing TBSV p33 replication protein.
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S4 Fig. Confocal laser microscopic images of CIRV-infected plant cells expressing CIRV p36 replication protein.
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S5 Fig. Confocal laser microscopic images of uninfected plant cells.
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S6 Fig. Enhanced co-purification of Osh6p with p33/p92 replication proteins from actin mutant yeast.
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S1 Video. 3D Super Resolution laser microscopic image of a yeast cell.