A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection Don B. Gammon1, Sophie Duraffour2, Daniel K. Rozelle3, Heidi Hehnly4, Rita Sharma1,9, Michael E. Sparks5, Cara C. West6, Ying Chen1, James J. Moresco7, Graciela Andrei2, John H. Connor3, Darryl Conte Jr1., Dawn E. Gundersen-Rindal5, William L. Marshall6,8#, John R. Yates III7, Neal Silverman6 and Craig C. Mello1,9,*. 1University of Massachusetts Medical School, RNA Therapeutics Institute, Worcester, MA, USA. 2Rega Institute for Medical Research, Leuven, Belgium. 3Boston University, Department of Microbiology, Boston, MA, USA. 4University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA, USA. 5United States Department of Agriculture, Agricultural Research Service, Beltsville, MD, USA. 6University of Massachusetts Medical School, Department of Medicine, Worcester, MA, USA. 7The Scripps Research Institute, Department of Chemical Physiology, La Jolla, CA, USA. 8Merck Research Laboratories, Boston, MA, USA. #Current Address. 9Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA. *Corresponding author: Please address all correspondence to Dr. Craig Mello at the University of Massachusetts Medical School. Email: [email protected]. Telephone: 508-856-1602. 1 1 Abstract 2 Virus-host interactions drive a remarkable diversity of immune responses and 3 countermeasures. We found that two RNA viruses with broad host ranges, vesicular 4 stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their 5 replication after entry into Lepidopteran cells. This restriction is overcome when cells 6 are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi 7 screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin- 8 proteasome pathways restrict RNA virus infection. Surprisingly, a highly-conserved, 9 uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We 10 show that A51R is also critical for VACV replication in vertebrate cells and for 11 pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host 12 microtubules and also associates with ubiquitin. We show that A51R promotes viral 13 protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for 14 destruction. Importantly, our studies reveal exciting new opportunities to study virus- 15 host interactions in experimentally-tractable Lepidopteran systems. 2 16 INTRODUCTION 17 Viruses represent a constantly evolving challenge to the fitness and survival of 18 their cellular hosts. Thus, not surprisingly, investigations into virus-host interactions 19 have produced important and fundamental new insights into both cellular and 20 pathophysiology (Panda & Cherry, 2012). Invertebrate model organisms have proven 21 useful in elucidating a wide range of host responses to infection and because many of 22 these responses are well conserved, studies in model organisms are often directly 23 relevant to human health (Moser, Jones, Thompson, Coyne, & Cherry, 2010; Moy et al., 24 2014; Panda & Cherry, 2012). Notably, studies of invertebrate antiviral RNA 25 interference (RNAi) pathways (Fire et al., 1998; Zhou & Rana, 2013) have produced 26 powerful tools for probing and manipulating gene function, with potential utility for direct 27 therapeutic intervention (Blake, Bokhari, & McMillan, 2012). 28 Relatively small genomes, well-defined genetics, and efficient RNAi pathways 29 make insect models attractive systems in which to study virus-host interplay (Cherry, 30 2011; Moser et al., 2010). Dipteran organisms, such as Drosophila melanogaster, have 31 been the primary focus of virus-host studies in invertebrates and Drosophila RNAi 32 screens have greatly enhanced our understanding of how eukaryotic host factors can 33 promote or inhibit virus replication (Cherry, 2011). These studies have almost 34 exclusively focused on RNA viruses (Xu & Cherry, 2014). In contrast to Dipterans, most 35 virus-host studies in the order Lepidoptera (moths and butterflies) have focused on DNA 36 viruses, particularly baculoviruses (Ikeda, Yamada, Hamajima, & Kobayashi, 2013). 37 These studies have provided key insights into highly conserved mechanisms by which 38 Lepidopterans combat DNA virus infection (Ikeda et al., 2013). Thus, while 3 39 Lepidopterans provide a relevant model for studying DNA virus-host interaction they 40 have not previously been used to probe restrictions to RNA virus replication. 41 The gypsy moth (Lymantria dispar) has been one of the most prolific North 42 American hardwood forest pests since its accidental release in the late 1800’s (Sparks, 43 Blackburn, Kuhar, & Gundersen-Rindal, 2013). Exploring the susceptibility and 44 responses of L. dispar and other Lepidopterans to virus infection is of particular 45 importance in designing new and effective virus-based biocontrol strategies to minimize 46 the devastating economic impact these species continue to have on the forest industry 47 (Sparks et al., 2013). L. dispar-derived cell lines are susceptible to a wide variety of 48 invertebrate DNA viruses, and as such, they are often used in virus-host studies 49 (Sparks & Gundersen-Rindal, 2011). Interestingly, L. dispar-derived LD652 cells can 50 also support a limited infection by vaccinia virus (VACV), a vertebrate poxvirus encoding 51 a large dsDNA genome (Li, Yuan, & Moyer, 1998). During infection of LD652 cells, 52 VACV undergoes early gene expression, DNA replication and late gene expression, but 53 the infection is abortive due to a defect in one or more steps of virion morphogenesis (Li 54 et al., 1998). VACV entry and early gene expression have also been documented in 55 Drosophila cells, however viral DNA replication and subsequent late gene expression 56 were not detected, indicating that VACV replication is blocked earlier in its life cycle in 57 Drosophila cells than in LD652 cells (Moser et al., 2010). Despite these limitations, 58 RNAi screening of VACV-infected Drosophila cells has identified multiple host factors 59 required for VACV entry in eukaryotic hosts (Moser et al., 2010). Thus, the LD652 cell 60 culture system provides a unique model in which to explore multiple aspects of 61 vertebrate DNA virus biology, including basic replication strategies and suppression of 4 62 host immune pathways by viral proteins. 63 The extent of RNA virus studies in Lepidoptera is limited compared to DNA virus 64 studies and largely restricted to non-enveloped dsRNA and (+)-sense ssRNA viruses 65 such as cypoviruses (Hill, Booth, Stuart, & Mertens, 1999), iflaviruses (van Oers, 2010) 66 and tetraviruses (Short & Dorrington, 2012). These viruses only infect invertebrate hosts 67 and several cannot productively replicate in cultured cells (Short & Dorrington, 2012). 68 Furthermore, to our knowledge, (-)-sense ssRNA viruses have not been previously 69 reported to productively infect Lepidopteran hosts. A new model system for studying 70 RNA viruses in Lepidopteran hosts may be useful in the design of new biocontrol agents 71 for pest species and improve our understanding of RNA virus-induced disease in 72 vertebrates. 73 Here we explore RNA virus-Lepidopteran host interactions by infecting LD652 74 cells with the (-)-sense ssRNA vesicular stomatitis virus (VSV) or the (+)-sense ssRNA 75 Sindbis virus (SINV), both of which replicate in a wide range of invertebrate and 76 vertebrate hosts (Letchworth, Rodriguez, & Del cbarrera, 1999; Xiong et al., 1989). We 77 unexpectedly found that LD652 cells restrict both VSV and SINV replication after virus 78 entry. Using RNAi to knock down the expression of candidate L. dispar antiviral 79 immunity factors, we show that specific RNAi and innate immune pathway components 80 restrict RNA virus replication. We also uncover a role for the Lepidopteran ubiquitin- 81 proteasome system (UPS) in restricting RNA virus replication. Surprisingly, co-infection 82 with VACV strongly suppressed this restriction, suggesting that VACV encodes one or 83 more factors that promote RNA virus replication. Using RNAi and genetic techniques, 84 we found that the highly conserved, and previously uncharacterized, VACV A51R gene 5 85 product is sufficient to alleviate the LD652 cell restriction to VSV and SINV replication. 86 Interestingly, A51R formed aggregate- and filament-like structures that colocalize with 87 microtubules (MTs) and protected MTs from depolymerization. Using alanine 88 mutagenesis, we further show that an A51R point mutant with reduced RNA virus 89 rescue ability still forms filamentous structures and stabilizes MTs, suggesting that 90 A51R functions, in addition to MT stabilization, are required for disarming Lepidopteran 91 antiviral immunity. Using mass spectrometry-based techniques, we found that A51R co- 92 immunoprecipitates with several host proteins, including ubiquitin (Ub). Using 93 radiolabeling and immunoblotting, we show that A51R does not affect viral mRNA 94 translation rates but does promote virus protein stability, possibly by inhibiting Ub- 95 dependent host targeting of viral proteins for degradation. Importantly, we show that 96 A51R is also required for efficient replication of VACV in vertebrate cells and for 97 pathogenesis in mice, indicating that A51R is a VACV virulence factor. Collectively, our 98 findings demonstrate the utility of Lepidopteran systems for the study of RNA- and DNA- 99 virus host interactions and shed light on how this