Orsay, Santeuil and Le Blanc Viruses Primarily Infect Intestinal Cells in Caenorhabditis Nematodes
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Virology 448 (2014) 255–264 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Orsay, Santeuil and Le Blanc viruses primarily infect intestinal cells in Caenorhabditis nematodes Carl J. Franz a, Hilary Renshaw a, Lise Frezal b, Yanfang Jiang a, Marie-Anne Félix b, David Wang a,n a Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, 660 S. Euclid Avenue, St. Louis, MO, USA b Institute of Biology of the Ecole Normale Supérieure, CNRS UMR8197, Inserm U1024, Paris, France article info abstract Article history: The discoveries of Orsay, Santeuil and Le Blanc viruses, three viruses infecting either Caenorhabditis Received 20 September 2013 elegans or its relative Caenorhabditis briggsae, enable the study of virus–host interactions using natural Accepted 26 September 2013 pathogens of these two well-established model organisms. We characterized the tissue tropism of Available online 1 November 2013 infection in Caenorhabditis nematodes by these viruses. Using immunofluorescence assays targeting Keywords: proteins from each of the viruses, and in situ hybridization, we demonstrate viral proteins and RNAs Tissue tropism localize to intestinal cells in larval stage Caenorhabditis nematodes. Viral proteins were detected in one to Nematode virology six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral fl Immuno uorescence proteins were detected as early as 6 h post-infection. The RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns. Collectively, these observa- tions provide the first experimental insights into viral protein expression in any nematode host, and broaden our understanding of viral infection in Caenorhabditis nematodes. & 2013 Elsevier Inc. All rights reserved. Introduction subsequent transmission. Previous studies of viruses in C. elegans have relied upon nematode cell culture systems (Wilkins et al., The first viruses capable of naturally infecting Caenorhabditis 2005; Schott et al., 2005), a transgenic virus replicon (Lu et al., nematodes were recently described (Felix et al., 2011; Franz et al., 2005) or artificial PEG-mediated conditions for “infection” (Liu 2012). Orsay virus, which was discovered in the wild C. elegans et al., 2006). These studies identified RNAi and programmed isolate JU1580, can infect multiple strains of C. elegans including cell death genes as antiviral pathways. Dicer-related helicase-1 the laboratory reference strain N2 (Felix et al., 2011). Santeuil virus (DRH-1), an ortholog of the human RIG-I receptor gene, was was discovered in the JU1264 wild C. briggsae isolate, and a third identified as an integral member of the anti-viral RNAi pathway in virus, Le Blanc virus, was recently discovered in the JU1498 wild C. C. elegans expressing the transgenic Flock house virus (FHV) (Lu et al., briggsae isolate (Felix et al., 2011; Franz et al., 2012). Studies of 2009). Furthermore, the virus-derived small interfering RNAs (viR- Orsay virus infection of C. elegans demonstrated that defined NAs) generated in response to a Flock house virus replicon were mutations in RNAi pathway genes, such as the rde-1 mutant in shown to be transgenerationally inherited for up to at least three the N2 background, lead to 50–100 fold increased viral RNA generations (Rechavi et al., 2011). More recently, the viRNAs derived accumulation compared to wild type N2. The wild nematode from a FHV replicon modified to express a fragment of the C. elegans isolate JU1580, which is partially defective for RNAi, is as suscep- gene, unc-22, demonstrated virus-induced silencing of unc-22 cellular tible to Orsay infection as the rde-1 mutant strain based on qRT- transcripts in C. elegans (Guo et al., 2012). PCR (Felix et al., 2011). Phylogenetic analysis of these three nematode viruses indicates The discoveries of these viruses enable, for the first time, the that all three are most closely related to each other and distantly study of virus–host interactions in Caenorhabditis nematodes using related to viruses in the family Nodaviridae. While the formal natural viruses fully competent for infection, replication and classification of Orsay, Le Blanc, and Santeuil viruses remains to be determined by the International Committee for the Taxonomy of Viruses (ICTV), comparisons to their closest characterized relatives, n Correspondence to: Washington University in St. Louis School of Medicine, the nodaviruses, are instructive and may provide useful paradigms. Campus box 8230, 660 S. Euclid Ave., St. Louis, MO 63110 USA. Fax: þ1 314 362 1232. Nodaviruses are positive-sense, single stranded RNA viruses E-mail address: [email protected] (D. Wang). isolated from a broad range of insect and fish hosts. Nodavirus 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.09.024 256 C.J. Franz et al. / Virology 448 (2014) 255–264 genomes are comprised of two single segments of 5′ capped, non- future exploitation of the Caenorhabditis nematode model of virus polyadenylated RNA (Newman and Brown, 1976). The RNA1 infection. segment of nodaviruses ranges in size from 3.0 to 3.2 kb and encodes the viral RNA-dependent RNA polymerase (RdRP). The RNA2 segment is 1.4 kb and encodes the capsid protein. Orsay, Results Le Blanc, and Santeuil viruses share some similarity in genomic organization with nodaviruses but differ due to the presence of an Orsay, Santeuil and Le Blanc viruses share a common tissue tropism additional ORF in the RNA2 segment (Felix et al., 2011). In addition, there is currently no computational or experimental evidence that Our previous work using light and electron microscopy sug- the genomes of Orsay, Le Blanc, or Santeuil viruses encode a B2 gested that C. elegans intestinal cells are structurally affected upon protein, a known suppressor of RNAi translated from the sub- Orsay virus infection (Felix et al., 2011). Using in situ hybridization genomic RNA3 strand in nodaviruses (Felix et al., 2011; Li et al., on adult animals, the Orsay virus RNA1 segment can be detected in 2002). Furthermore, a recent study suggests that the proteins intestinal cells and possibly cells of the somatic gonad (Felix et al., encoded by the Orsay virus RNA2 strand do not have RNAi 2011). To better define the cellular tropism of Orsay virus infection suppression properties (Guo and Lu, 2013). in C. elegans, we raised antibodies against peptides derived from Nodaviruses are classified into two distinct genera: alphano- the Orsay virus RdRP and capsid proteins for use in immunofluor- daviruses which infect insects, and betanodaviruses which pri- escence assays (IFA). These antibodies were used to assess Orsay marily infect fish. While there have been few reports describing infection of the nematode strains JU1580, N2 and WM27 (rde-1 the tissue tropism of alphanodaviruses, Flock House virus (FHV), mutant). IFA of Orsay virus-infected C. elegans adult animals the prototype of alphanodaviruses, infects the midgut, head, fat yielded high levels of non-specific staining for all three nematode body, and salivary glands in mosquitoes (Aedes aegypti) based on strains rendering it difficult to interpret (data not shown). By immunofluorescence assays (IFA) (Dasgupta et al., 2003). In cell contrast, Orsay virus-infected but not mock-infected rde-1 mutant culture, FHV can replicate in insect, mammalian, plant, and yeast larvae displayed a strong specific staining pattern of discrete cells cells (Ball, 1992; Gallagher et al., 1983; Selling et al., 1990; Price with both the anti-Orsay RdRP (Fig. 1) and the anti-Orsay capsid et al., 1996); furthermore, a FHV genomic replicon has been (Fig. 2) antibodies. Some non-specific staining was observed in the established in C. elegans (Lu et al., 2005). Among the betanoda- anterior region, near the mouth and pharynx of a given animal, in viruses, Striped jack nervous necrosis virus has been detected by both mock-infected (Figs. 1A and 2A) and infected animals (Figs. 1B IFA in the central nervous system and retina of infected larval and 2B). The wild C. elegans isolate JU1580 and rde-1 mutant striped jack (Pseudocaranx dentex)(Iwamoto et al., 2005). Simi- animals in the N2 background, which are equally sensitive to larly, Atlantic halibut nodavirus capsid protein was detected by Orsay infection as measured by qRT-PCR (Felix et al., 2011), yielded immunohistochemistry (IHC) in the brain, spinal cord, and intes- the same general staining pattern (Figs. 1–3 and data not shown). tine of larval Atlantic halibut (Hippoglossus hippoglossus)(Grotmol The laboratory wild type reference N2 strain, which is less et al., 1999) and in the brain and retina of infected adult Atlantic permissive to Orsay virus and accumulates 50–100 fold less viral cod (Gadus morhua)(Korsnes et al., 2009). The capsid protein of RNA than the JU1580 isolate (Felix et al., 2011) did not yield any the unclassified shrimp nodavirus, Macrobrachium rosenbergii animals with positive staining cells (n¼100 animals per experi- nodavirus, was observed by IHC in a variety of tissues that include ment, 15 independent experiments). the muscles, nerve cord, gill, heart, loose connective tissue, and Many infected rde-1 mutant animals showed a single positive hepatopancreas of infected shrimp (Macrobrachium rosenbergii) staining cell (Figs. 1B and C; 2B and C). In addition, animals with (Wangman et al., 2012). Thus, nodaviruses have a broad range of more than one RdRP-positive (Fig. 1D–F) or capsid-positive tissue tropisms. (Fig. 2D and E) cell were observed, with a maximum of six positive The discovery of viruses that naturally infect Caenorhabditis cells detected. In most cases, the positive cells were adjacent to nematodes provides a unique opportunity to explore virus–host each other (Figs. 1D–F and 2E); however, some non-adjacent interactions in the context of a well-established model organism. positive cells were observed (Fig.