Sasvari et al., 2014 1 2 3 Tombusvirus-yeast interactions identify conserved 4 cell-intrinsic viral restriction factors 5 6 a# a# a* 7 Zsuzsanna Sasvari , Paulina Alatriste Gonzalez and Peter D. Nagy 8 9 10 11 aDepartment of Plant Pathology, University of Kentucky, Lexington, KY 12 # 13 These authors contributed equally to this work. 14 15 *To whom correspondence should be addressed at the Department of Plant Pathology, 16 University of Kentucky, 201F Plant Science Building, Lexington, KY 40546; Tel: (859) 17 2180726; Fax: (859) 323-1961; E-Mail: [email protected] 18 19 20 1 Sasvari et al., 2014 1 Abstract: To combat viral infections, plants possess innate and adaptive immune 2 pathways, such as RNA silencing, R gene and recessive gene-mediated resistance 3 mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF) 4 are also involved in limiting plant virus replication. This review discusses novel CIRFs with 5 antiviral functions, many of them RNA-binding proteins or affecting the RNA binding 6 activities of viral replication proteins. The CIRFs against tombusviruses have been 7 identified in yeast (Saccharomyces cerevisiae), which is developed as an advanced model 8 organism. Grouping of the identified CIRFs based on their known cellular functions and 9 subcellular localization in yeast reveals that TBSV replication is limited by a wide variety 10 of host gene functions. Yeast proteins with the highest connectivity in the network map 11 include the well-characterized Xrn1p 5’-3’ exoribonuclease, Act1p actin protein and Cse4p 12 centromere protein. The protein network map also reveals an important interplay between 13 the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key 14 roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of 15 selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, 16 single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. 17 These restriction factors frequently target the RNA-binding region in the viral replication 18 proteins, thus interfering with the recruitment of the viral RNA for replication and the 19 assembly of the membrane-bound viral replicase. Although many of the characterized 20 CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function 21 as “guardians” of the cellular Hsp70 chaperone system, which is subverted efficiently by 22 TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones. 23 2 Sasvari et al., 2014 1 Key words: RNA-binding host proteins, inhibition of viral RNA recruitment, plant resistance; 2 innate immunity; antiviral response; cell-intrinsic restriction factor; inhibition of virus 3 replication; cellular factor; viral replicase complex; host factors, genome-wide screens; RNA- 4 protein interaction, yeast as a host, protein-protein interaction; protein network; Arabidopsis. 5 6 3 Sasvari et al., 2014 1 Viruses with RNA genomes are widespread pathogens of plants, animals and humans. 2 RNA viruses have small genomes with limited coding potential, yet they replicate efficiently in 3 the infected host cells by co-opting numerous host proteins and subverting subcellular 4 membranes to build replication factories/organelles [1-4]. During the infection process, RNA 5 viruses rewire many cellular pathways that render the cells dramatically different from 6 uninfected cells. However, cells have also developed antiviral strategies to limit viral infections 7 and host organisms are in constant evolutionary battle with viruses, leading to several layers of 8 antiviral responses from the host and emergence of novel suppressors /effectors by viruses. 9 Plants do not have the immune system of mammals or the potent interferon response, yet 10 they possess innate immune pathways that provide non-specific and immediate response against 11 bacteria, fungi, and viruses. In plants, the innate immune response involves pathogen-associated 12 molecular pattern-triggered immunity (PTI), effector-triggered immunity (ETI), and different 13 protein kinases that perform surveillance, systemic signalling and chromosomal changes [5-7]. 14 Adaptive immunity in plants is an inducible defense system that responds to environmental cues. 15 Examples for adaptive immunity include the local hypersensitive response (HR) and systemic 16 acquired resistance (SAR) response, which causes resistance at the whole-plant level [8, 9]. 17 The most potent adaptive immune response against plant viruses is based on RNA 18 interference/RNA silencing (also called post-transcriptional gene silencing, PTGS), when the 19 accumulating viral dsRNA formed during positive-stranded (+)RNA virus replication [10] 20 induces the antiviral RNA silencing response [11-13]. Plant viruses counteract the RNA 21 silencing pathways by using viral suppressors [12, 14]. Another type of innate resistance 22 mechanism against plant viruses is based on R genes (dominant resistance), such as the N gene 23 of tobacco against Tobacco mosaic virus (TMV) [15, 16]. The N gene is a TIR-NB-LRR 4 Sasvari et al., 2014 1 receptor, which recognizes the TMV helicase protein to induce plant innate immunity [17]. A 2 less well-understood innate resistance mechanism against plant viruses is based on recessive 3 resistance genes. This type of innate immunity is usually based on recessive mutation(s) in host 4 genes that are co-opted by the virus, but could no longer support the need of the virus during 5 infection due to the mutations rendering the host protein “antiviral” [6, 18]. In spite of our 6 growing knowledge on the innate immune responses in plants, it is likely that additional, not yet 7 identified, cell-intrinsic restriction factors (CIRFs) are also involved in limiting plant virus 8 infections. This review discusses the genome-wide identification and detailed characterization of 9 CIRFs with antiviral functions based on the plant-infecting tombusviruses. 10 11 1. Identification of novel cell-intrinsic restriction factors during viral infection based 12 on yeast. Most RNA viruses of plants have small RNA genomes coding for 5-15 viral proteins 13 only that are insufficient to support viral replication without subverted host factors, subcellular 14 membranes and cellular metabolites, such as ribonucleotides and amino acids [1, 19]. The 15 genome-size limitation and the biology of the virus makes RNA viruses much more dependent 16 on the host cells in comparison with other plant pathogens, such as fungi and bacteria. In 17 addition, the entire infectious cycle of plant RNA viruses takes place inside the infected cells, 18 thus making the viral RNAs more accessible to cellular antiviral factors that could destroy viral 19 RNAs and viral proteins in the cytosol. However, the limited number of effectors expressed by 20 viruses, their intracellular presence and “the stealth mode” of viral activities also mean that 21 viruses could more readily avoid recognition by the host in comparison with other pathogens. 22 Therefore, plant cells might need to deploy numerous CIRFs against viruses. The identification 5 Sasvari et al., 2014 1 of the putative CIRFs could be accelerated by unbiased genome-wide screens in host plants, 2 which have not yet been accomplished. 3 Although Saccharomyces cerevisiae (baker yeast) lacks well-known antiviral pathways, 4 such as the adaptive immune system of mammals, the interferon response or other innate 5 immunity systems including the RNAi pathway, yeast cells can still protect themselves against 6 viruses [20, 21]. To discover if yeast codes for CIRFs against viruses, high throughput screens 7 using yeast genomic libraries have been performed based on small plant viruses, such as Brome 8 mosaic virus (BMV) and Tomato bushy stunt virus (TBSV) [22]. 9 Yeast is a powerful surrogate host for some plant viruses to help researchers screen for 10 CIRFs. This is due to the small genome (only ~6,000 genes, with 75% of genes have assigned 11 functions and ~50% of genes have human and/or plant orthologs), and available extensive strain 12 libraries [23]. Moreover, yeast not only facilitates genome-wide studies, it is also helpful for 13 validation of the identified cellular factors and dissection of their functions to limit viral 14 replication, as discussed below. 15 Although this review focuses on the results obtained mainly with TBSV, which is among 16 the most intensively studied plant positive-strand (+)RNA viruses, CIRFs have also been 17 identified for BMV and Flock house virus (FHV) an insect virus, based on yeast screens, as well 18 [24-26]. Therefore, it is likely that detailed studies on CIRFs will expand to BMV, FHV and 19 possibly more viruses using yeast as a model host. 20 21 1.1. High-throughput genome-wide screens in yeast for systematic identification of 22 cell-intrinsic restriction factors limiting viral replication. The most efficient approach to 23 identify CIRFs is based on unbiased genome-wide screens that measure the level of virus 6 Sasvari et al., 2014 1 replication [1, 22, 25, 27-29]. However, this approach is not yet straightforward to perform with 2 plants that have a large number of genes and show high level of gene- (or functional-) 3 redundancy that makes it challenging for scientists to identify CIRFs. The Ahlquist lab has 4 pioneered the use of yeast as a viral host and performed a low throughput genetic mutagenesis 5 screen and systematic screens to identify host genes affecting virus replication [24, 26, 30]. The 6 most extensive genome-wide screens based on yeast libraries were performed with TBSV [1, 22, 7 29, 31-34]. These included knock-out and knock-down libraries and a temperature-sensitive (ts) 8 mutant library of yeast [35] for TBSV replication studies, leading to the identification of 73 yeast 9 genes acting as CIRFs against viral infection [31-34, 36, 37]. 10 Grouping of the identified CIRFs based on their known cellular functions and subcellular 11 localization in yeast reveals that TBSV replication is limited by a wide variety of gene functions 12 (Table 1).