Host factors involved in RNA replication of Dianthovirus( Title Dissertation_全文 ) Author(s) Hyodo, Kiwamu Citation 京都大学 Issue Date 2014-03-24 URL https://doi.org/10.14989/doctor.k18333 Right 許諾条件により本文は2015-03-24に公開 Type Thesis or Dissertation Textversion ETD Kyoto University Host factors involved in RNA replication of Dianthovirus Kiwamu Hyodo 2014 Contents General Introduction 1 ChapterI 6 Identification of amino acids in auxiliary replicase protein p27 critical for its RNA-binding activity and the assembly of the replicase complex in Red clover necrotic mosaic virus ChapterII 34 ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus ChapterIII 69 Functional analysis of phospholipase D and phosphatidic acid in a plant RNA virus replication References 91 Summary 107 Acknowledgements 110 General Introduction Viruses are obligate intracellular symbionts. Viral genomes consist of a single or double-stranded DNA or RNA encoding genetic information required for viral entry, replication and spread. Viruses are now thought to have been involved in evolution of organisms including animals and plants by symbiotic association (Roossinck 2005). Meanwhile, viruses have also been recognized as parasites that invade cells and hijack cellular machinery for their own purposes. In fact, viruses cause a large numbers of human diseases, including hepatic inflammation, immune deficiency syndrome, cancer, and so on. In addition, viruses are pathogenic agents that devastate cereals, vegetable and live stocks, which are essential to the viability of human-beings. As the world population continues to increase, more foods are needed for sustaining the population. To manage the food crisis, it is important to control viral infection in plants and live stocks. However, there is no established direct method to control viral diseases, in part because we do not fully understand how virus replicates in cells of plants and animals. Positive-strand RNA [(+)RNA] viruses are the most abundant in plant viruses, and include many economically important viruses in agriculture. Following entry into host cells, viral genomic RNAs are released from virions into the cytoplasm of the host cells and act as templates for translation to produce replication proteins using the host’s translation machinery (Dreher and Miller 2006; Simon and Miller 2013). The replication proteins recruit genomic RNA together with host proteins to form the viral replication complex (VRC). The VRC synthesizes a complementary negative-strand RNA [(–)RNA] using the original genomic RNA as a template. The (–)RNA is then used as a template to synthesize many new (+)RNAs that undergo additional rounds of translation and replication, or move to adjacent cells, or are encapsidated into virions (Buck 1996; Nagy and Pogany 2012). Although (+)RNA viruses have a limited coding capacity and code for only several genes, RNA viruses perform many tasks (e.g., synthesis of viral proteins and RNAs, 1 regulation of RNA replication and gene expression, escape from antiviral responses, and cell-to-cell and long distance movement). To accomplish these tasks, viruses seem to have acquired the ability to use host-derived proteins, membranes, lipids, and metabolites, and to exploit or rewire cellular trafficking pathways during evolution. All characterized eukaryotic (+)RNA viruses assemble VRCs, which contain both viral and host proteins, on intracellular membranes (Ahlquist et al. 2003; den Boon et al. 2010; den Boon and Ahlquist 2010; Laliberté and Sanfaçon 2010; Miller and Krijnse-Locker 2008; Nagy and Pogany 2012). These membranes can be derived from various organelles such as the endoplasmic reticulum (ER), Golgi, mitochondria, chloroplasts, peroxisomes, vacuoles, as well as the plasma membranes. RNA replication mechanisms of positive-stranded RNA viruses have been extensively studied. However, many questions remain unanswered, for example: how do viral replication proteins recognize specifically viral genomic RNAs from a pool of host RNAs (mRNA, tRNA and rRNA), how do viruses establish VRCs, what host factors are involved in viral multiplication? To understand details of the replication mechanisms of positive-strand RNA viruses, I use Red clover necrotic mosaic virus (RCNMV) as a model virus to investigate RNA replication mechanisms. RCNMV has several interesting features not observed in other positive-strand RNA viruses (Okuno and Hiruki, 2013). RCNMV is a member of the family Tombusviridae and the genus Dianthovirus that includes Carnation ring spot virus (CRSV) as a type member, and Sweet clover necrotic mosaic virus (SCNMV) (Okuno and Hiruki, 2013). Dianthoviruses are taxonomically distinct from other viruses in Tombusviridae (e.g., TBSV) because of the bipartite nature of their single-stranded positive sense RNA genome. Virions of RCNMV are about 35 nm in size and composed of 180 copies of a 37 kDa coat protein (CP) (Okuno and Hiruki, 2013). The two RNA genomes RNA1 (3.9kb) and RNA2 (1.45kb) (Gould et al., 1981; Hiruki, 1987; Okuno et al., 1983), lack both cap structure at the 5! end and poly(A) tail at the 3! end (Lommel et al., 1988; Mizumoto et al., 2003). RNA1 and 2 RNA2 share fundamentally no homology, except for the first seven nucleotides at the 5! ends and two stem-loop structures at the 3! ends in both genomic RNAs. RNA1 encodes RNA replicase components, a 27-kDa protein (p27) and its N-terminally overlapping 88-kDa RNA-dependent RNA polymerase (RdRP) (p88pol). p88pol produced by -1 frameshifting (Kim and Lommel 1994, 1998). Both p27 and p88pol are required for replication of RNA1 and RNA2 in plants or protoplasts (Takeda et al., 2005; Okamoto et al., 2008; Mine et al., 2010b). p27 and p88pol form 480 kDa RNA replication complex in RCNMV-infected plants (Mine et al., 2010b). This 480 kDa complex retained RdRp activity in vitro (Mine et al., 2010b). RNA1 also encodes a 37-kDa coat protein (CP) that is expressed from subgenomic RNA (CP sgRNA) (Tatsuta et al., 2005; Zavriev et al., 1996). Transcription of CP sgRNA requires intermolecular interaction between RNA1 and RNA2 (Sit et al., 1998; Tatsuta et al., 2005). RNA1 alone can replicate in a single cell, but can not move to neighboring cells without 35-kDa movement protein (MP) encoded by RNA2 (Lommel et al., 1988; Xiong et al., 1993; Kaido et al., 2009). Mechanisms of translation and RNA replication differ between RNA1 and RNA2. Cap-independent translation of RNA1 is mainly mediated by the translation-enhancer element of dianthovirus RNA1 (3!TE-DR1) that resides between 3596 nt and 3732 nt in the 3!-untranslated region (UTR) (Mizumoto et al., 2003). On the other hand, RNA2 does not have such translational enhancer elements as 3!-TE-DR1. Cap-independent translation of RNA2 is coupled with RNA replication, and, therefore, cis-acting RNA elements required for RNA replication are also essential for translation of RNA2 (Mizumoto et al., 2006). Replication mechanisms are also different between RNA1 and RNA2. Replication proteins were required in cis for the replication of RNA1, whereas RNA2 can utilize replication proteins supplied in trans for its replication (Takeda et al., 2005; Okamoto et al., 2008). RCNMV RNA replication is also required for counter-defence against RNA interference (RNAi), which is induced by double-strand RNA (dsRNA) and is involved in antiviral defense (Takeda et al., 2005). Host factors involved in (+)RNA plant virus replication have been identified using 3 several approaches. Genome-wide screening of Saccharomyces cerevisiae led to the identification of up to 130 genes that affect the replication of Tomato bushy stunt virus (TBSV) (Jiang et al. 2006; Panavas et al. 2005) or Brome mosaic virus (BMV) (Gancarz et al. 2011; Kushner et al. 2003) and up to 30 genes that affect TBSV RNA recombination (Serviene et al. 2005, 2006). Proteome-wide overexpression screening in S. cerevisiae also identified numerous host proteins that affect TBSV replication (Shah Nawaz-ul-Rehman et al. 2012). Mass spectrometric analysis of cellular proteins coimmunopurified with viral replication proteins has identified the host proteins that are part of the VRC or that interact with the viral replication proteins in several viruses, including Tomato mosaic virus (ToMV) (Nishikiori et al. 2006), TBSV (Serva and Nagy 2006), Potyviruses (Dufresne et al. 2008; Hafrén et al. 2010), and RCNMV (Mine et al. 2010b). Mass spectrometric analysis has also been used to identify host proteins that interact with the viral RNA of RCNMV (Iwakawa et al. 2012), Bamboo mosaic virus (BaMV) (Huang et al. 2012; Lin et al. 2007; Prasanth et al. 2011), and ToMV (Fujisaki and Ishikawa 2008). Protein microarray analysis led to the identification of host proteins that interact directly with the replication proteins of TBSV or with the viral RNAs of TBSV and BMV (Li et al. 2008, 2009; Zhu et al. 2007). Microarray analysis of host gene expression during viral infection and subsequent functional analysis has also been used to identify host factors involved in virus replication (Chen et al. 2013a, b). However, the functions of most host factors that have been identified using such approaches remain unknown. To reveal the RNA replication mechanisms, I investigated the functions of p27 replication protein. Using a viral translation/replication system containing cytoplasmic extracts prepared from tobacco BY-2 cells (BYL) (Komoda et al., 2004), which is a powerful tool to investigate individual determinants of the replication process of positive-strand RNA viruses, I revealed that p27 has multiple functions during RNA replication (Chapter I). Moreover, I identified the host proteins that interact with RCNMV replication proteins and revealed the importance of host membrane trafficking 4 pathway (Chapter II) and specific phospholipid (Chapter III) in viral RNA replication. Understanding the roles of host proteins as well as viral factors in viral replication provides information about the molecular pathways exploited by the virus and further targets that could be pursued in the development of antiviral strategies against existing and emerging plant virus diseases.