Date Published: February 10, 2016
Publisher: Public Library of Science
Author(s): Muhammad Shah Nawaz-ul-Rehman, K. Reddisiva Prasanth, Kai Xu, Zsuzsanna Sasvari, Nikolay Kovalev, Isabel Fernández de Castro Martín, Daniel Barajas, Cristina Risco, Peter D. Nagy, Aiming Wang.
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.
Plus-stranded (+)RNA viruses, which are important pathogens of plants, animals and humans, co-opt a number of host-coded proteins and lipids to facilitate the replication process [1–6]. These viruses also remodel host membranes and alter host cellular pathways to take advantage of host resources and to avoid recognition by host antiviral defenses. Characterization of an increasing number of host factors involved in (+)RNA virus replication has already revealed intriguing and complex interactions between various viruses and their hosts. Functional studies with selected host proteins have revealed a plethora of activities preformed by these host proteins during RNA virus infections [1,3,7–11]. In spite of the intensive efforts, our current cataloging of host factors is still far from complete and our current knowledge on the role of the identified host factors is incomplete.
Tombusvirus replication involves the assembly of hundreds-to-thousands of VRCs in infected cells that leads to robust viral RNA synthesis [45,71]. The VRCs are clustered in large viral-induced compartments, called replication organelles . The formation of individual VRCs, housed in spherule-like membranous structures, requires p33 and p92 replication proteins, the viral (+)RNA and 15–20 co-opted host proteins, lipids, such as sterols and phospholipids, and likely metabolites [3,16,43]. TBSV replication also depends on MCSs to transfer sterols to the sites of replication, which is the peroxisomal membrane for TBSV and mitochondrial outer membrane for CIRV . How can all these viral and cellular components come together at the sites of replication and facilitate the formation of large replication organelles containing relatively stable membranous VRCs inside the cytoplasm? Based on the current work, it seems that the actin network plays a key role in this critical viral process. Accordingly, the large p33 containing replication structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells (Figs 8–10). Therefore, these actin filaments are likely involved in VRC assembly and the formation of large viral replication organelles containing many individual VRCs. Indeed, expression of dominant negative mutant of myosin XI-K motor protein, which is involved in actin-based intracellular cargo movement [72,73], strongly inhibited tombusvirus accumulation in N. benthamiana leaves, supporting the functional role the actin network in tombusvirus replication (Fig 6B).