Orsay, Santeuil and Le Blanc Viruses Primarily Infect Intestinal Cells in Caenorhabditis Nematodes

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Virology 448 (2014) 255–264

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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 observations 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. 2D). However, this field is currently in its infancy; there is little known In many cases both antibodies stained the same cell in infected about the fundamental nature of virus infection in Caenorhabditis animals (Fig. 3A). In other instances we observed cells that were nematodes, or in any nematode for that matter. Despite the only positive for the anti-Orsay RdRP antibody (Fig. 3B). In tremendous diversity and abundance of nematodes worldwide addition, some animals had more cells that were positive with (Wilson, 2003), there are a limited number of studies that have the anti-Orsay RdRP antibody than with the anti-Orsay capsid reported on potential viruses that infect nematodes. Aside from antibody (Fig. 3C–E). However, we never observed capsid-positive the Caenorhabditis nematode infecting viruses, there have been and RdRP-negative cells. only a few electron microscope based studies that described virus- We next examined the localization of Le Blanc virus and like particles in parasitic nematodes (Poinar and Hess, 1977; Poinar Santeuil virus, two distantly-related viruses that infect wild et al., 1980; Zuckerman et al., 1973; Foor, 1972) and one recent RNA C. briggsae isolates (Felix et al., 2011; Franz et al., 2012). We sequencing study that detected multiple novel RNA virus genomes developed IFAs using antibodies against the RdRP proteins of Le in the soybean cyst nematode (Bekal et al., 2011). Detailed Blanc and Santeuil viruses. In mock-infected JU1498 and JU1264 molecular studies of virus infection in these other systems have animals, only non-specific staining near the mouth or pharynx not been described to date. was observed (Fig. 4A and D). In Le Blanc (Fig. 4B and C) and In this study, we developed robust immunofluorescence assays Santeuil (Fig. 4E and F) virus-infected animals, one or more clearly that detect Orsay, Santeuil and Le Blanc virus proteins, thereby delineated RdRP-positive staining cells could be detected, in establishing critical reagents for assessment of virus infection. patterns similar to that observed with Orsay virus (compare Application of these assays demonstrated that Orsay virus infec- Fig. 4 with Figs. 1 and 3). tion of C. elegans and Santeuil and Le Blanc virus infections of To validate the specificity of the positive staining of Orsay viral C. briggsae share a common primary tropism in intestinal cells. protein observed by IFA, the Orsay viral RNA1 and RNA2 segments Moreover, these studies present the first experimental data were stained by fluorescence in situ hybridization (FISH) in describing viral protein expression patterns and kinetics in the infected rde-1 mutant animals. The observed FISH staining pat- context of any nematode host, and they provide a foundation for terns for RNA1 (Fig. 5) and RNA2 (data not shown) were similar to C.J. Franz et al. / Virology 448 (2014) 255–264 257 those seen by IFA wherein one or more discrete cells in the central localizes just below the apical surface of intestinal cells (Francis portion of the animal stained positive. and Waterston, 1991). The Orsay capsid-positive cells also stained positive with the MH33 antibody, demonstrating that the infected cells were intestinal cells (Fig. 6). Moreover, the two antibodies Intestinal cell localization of Orsay virus appeared to colocalize or to be closely juxtaposed along the apical plasma membrane of capsid-positive cells (Fig. 6). We performed Based on previous FISH data in Orsay virus-infected adult similar co-staining with Le Blanc RdRP and MH33 and observed C. elegans (Felix et al., 2011), we hypothesized that the positive that the same cells stained positive with both antibodies (Supple- staining cells were intestinal. To explicitly test this hypothesis, mental Fig. S1). Orsay virus-infected rde-1 mutant animals were co-stained with anti-Orsay capsid and MH33, a well-characterized antibody that recognizes the intermediate filament 2 (IFB-2) protein and Kinetics of infection

To investigate the kinetics of Orsay virus protein expression, IFA and FISH were independently performed on the same populations of synchronized rde-1 mutant larvae ﬁxedat0,4,6,8,and12h following exposure to Orsay virus. For IFA, we performed co- staining experiments by simultaneously exposing animals to both anti-Orsay RdRP and anti-Orsay capsid antibodies.

Fig. 1. Immunofluorescence of Orsay virus-infected C. elegans using an anti-RdRP Fig. 2. Immunofluorescence of Orsay virus-infected C. elegans using an anti- antibody. Mock-infected (A) and Orsay virus-infected (B–F) rde-1 mutant larvae capsid antibody. Mock-infected (A) and Orsay virus-infected (B–E) rde-1 mutant stained with a polyclonal goat antibody against the Orsay virus RdRP. Nuclei were larvae stained with a polyclonal rabbit antibody against the Orsay virus capsid. counterstained with Hoechst (merge panels). Arrowheads demarcate specific Nuclei were counterstained with Hoechst (merge panels). Arrowheads demar- antibody staining in cells. NS: non-specific staining and H: head. Scale bars cate specific antibody staining in cells. NS: non-specific staining and H: head. represent 20 μm. Scale bars represent 20 μm. 258 C.J. Franz et al. / Virology 448 (2014) 255–264

Fig. 3. Double immunoﬂuorescence of Orsay virus-infected C. elegans using anti-RdRP and anti-capsid antibodies. Orsay virus-infected Ju1580 larvae (A–C) and rde-1 larvae (D and E) were simultaneously stained with polyclonal goat anti-RdRP and polyclonal rabbit anti-capsid antibodies. Nuclei were counterstained with Hoechst (merge panels). Scale bars represent 20 mm.

In three independent time course experiments, we scored the 2 cell positive, 14% 3 cell positive 12% cell positive and 13% 5 or number of animals with detectable levels of RdRP and/or capsid 6 cell positive. proteins out of 100 total animals per designated time point. At the In addition, we also quantified Orsay virus-infected rde-1 0 and 4 h time points, viral protein was not detected (Fig. 7). Orsay mutant animals that were positive by FISH. The Orsay RNA1 RdRP-positive cells were first observed at 6 hpi (31% of rde-1 segment was detected at 8 hpi in 74% of infected animals (data animals) and by 12 hpi, the average number of positive animals not shown). Most of the FISH-positive infected cells were located had increased to 76% (Fig. 7). Similarly, the capsid was also in the anterior half (98.4% at 8 hpi) of the animal. first detectable at 6 h and increased from 12% to 61% by 12 hpi (Fig. 7). At the 12 h time point, we further quantified the Orsay capsid and RdRP have distinct subcellular expression patterns distribution of the IFA staining patterns in terms of the average percentage of animals (n¼3 experiments, 100 animals per We further analyzed the subcellular expression of Orsay virus experiment) with 1, 2, 3, 4 or 5–6 cells that were positive for RdRP and capsid proteins using confocal microscopy. Both Orsay Orsay virus RdRP. We observed on average 33% 1 cell positive, 27% virus RdRP and capsid proteins were excluded from the nuclei (Fig. 8B, C.J. Franz et al. / Virology 448 (2014) 255–264 259

Fig. 5. Fluorescence in situ hybridization staining of Orsay viral RNA1 in C. elegans rde-1 mutant. Mock-infected (A) and Orsay virus-infected (B–E) rde-1 mutant larvae with RNA1 labeled by a Quasars 670 probe. Nuclei were counterstained with DAPI (merge panels). H: nuclei. Scale bars represent 20 µm.

we observed punctate staining patterns in the cytoplasm that were independent from the apical surface and vesicle-like structures (Figs. 6 and 8C).

Fig. 4. Immunofluorescence of Le Blanc virus or Santeuil virus-infected C. briggsae using anti-RdRP antibodies. Mock-infected (A) and Le Blanc virus-infected (B and C) JU1498 larvae were stained with a polyclonal rabbit antibody against Le Blanc virus Discussion RdRP. Mock-infected (D) and Santeuil virus-infected (E and F) JU1264 larvae were stained with a polyclonal rabbit antibody against the Santeuil virus RdRP. Nuclei The recent discovery of Orsay, Santeuil, and Le Blanc viruses as were counterstained with Hoechst (merge panels). Scale bars represent 20 μm. natural pathogens of Caenorhabditis nematodes provides new opportunities to explore virus–host interactions at the molecular, organismal and population levels. However, to further understand these interactions, it is necessary to gain a better understanding of n). Similarly, the Orsay RNA1 segment was also excluded from the the fundamental biological processes that occur during infection of nuclei (Fig. 5D, n). Furthermore, Orsay virus RdRP and capsid proteins Caenorhabditis nematodes. We determined that the three Caenor- had distinct subcellular localization patterns that appeared to be habditis nematode viruses share a common tissue tropism, namely mutually exclusive, as no colocalization was observed (Fig. 8). The localization primarily to intestinal cells in larval stage animals. In RdRP staining appeared throughout most of the cytoplasm. The capsid this process, we established the first immunofluorescence assays staining localized to the apical surface of the cell (Fig. 8BandC)and for detection of Orsay, Le Blanc and Santeuil virus proteins. We what appeared to be large vesicle-like structures (Fig. 8B). In addition, demonstrated that Orsay virus protein expression was detectable 260 C.J. Franz et al. / Virology 448 (2014) 255–264

Fig. 6. Intestinal cell localization of Orsay viral proteins in nematode host. Orsay virus-infected rde-1 larvae were stained with polyclonal rabbit anti-capsid and MH33 monoclonal mouse anti-IFB-2 antibodies and visualized by confocal microscopy. White boxes depict enlarged regions of interest in the panels directly below. Nuclei were counterstained with TO-PRO-3 (merge panel). a: capsid staining at the apical surface of intestinal cells and p: punctate capsid staining in the cytoplasm. Asterisk demarcates lack of MH33 staining. Scale bars represent 20 μm. in strains (JU1580 and rde-1 mutant) that have defects in the RNAi non-speciﬁc binding or other artifacts. In addition to co-staining pathway, but not in the RNAi-competent reference strain (N2). For with two antibodies that each target different viral proteins, we Orsay virus, we used multiple independent assays to ensure that also compared the patterns from the IFA to patterns obtained our observations were the result of speciﬁc staining and not due to using FISH. The similarity of the staining patterns of Orsay RNA C.J. Franz et al. / Virology 448 (2014) 255–264 261

Orsay Infection Time Course S2 cells infected with FHV, fluorescent-labeled capsid protein forms 100 densepatchesinthecytoplasm(Lanman et al., 2008). In some cells, RdRP 80 we observed dense punctate staining of the Orsay capsid. Further- Capsid more, Orsay virus capsid protein could often be detected near the 60 apical surface of intestinal cells. It is possible this reflects the site of viral assembly and/or egress. In some infected cells, we observed 40 diminished intermediate filament staining (MH33) in precisely the region where robust apical capsid staining was present (Fig. 6C, 20 asterisk). Reorganization of actin and intermediate filaments at the apical surface of the intestine has been reported in studies of the only Animals positive by IFA 0 other known intracellular pathogen of C. elegans, the microsporidium 0 4 6 8 12 N. parisii (Estes et al., 2011). To facilitate the exit of spores from Hours post infection intestinal cells, actin relocalizes from the apical to the basolateral surface, causing the formation of terminal web gaps in the inter- Fig. 7. Orsay infection time course in C. elegans rde-1 strain. For each time point, fi the number of animals that were IFA positive for RdRP or capsid was scored out mediate laments at the apical surface. Likewise, Orsay virus must of 100 animals total. The mean and standard deviation from three independent exit from the infected cell somehow, presumably into the intestinal infection time courses are plotted. lumen in order to effectively spread, and this observation raises the possibility that Orsay virus infection may somehow modulate orga- and the Orsay capsid and RdRP, wherein one or more cells of the nization of the apical surface of the intestinal cells. intestine yielded positive staining, demonstrated that the assays Using the IFA in the context of an infection time course, we we used were indeed specific; however, the possibility remains were able to define some basic parameters relevant to the virus that other tissue types may support low-level infection that is life cycle. Both viral capsid and RdRP were first detectable at 6 h below the threshold of detection by IFA or FISH. post-infection. This timing is comparable to nodaviruses such as The intestine is a primary organ in Caenorhabditis nematodes that BBV and FHV, which express detectable viral proteins in infected is composed of 20 cells in larvae and adults. It makes up approxi- Drosophila cells at 6 and 8 hpi, respectively (Friesen and Rueckert, mately one-third of the total somatic body mass and is vital for 1981; Gallagher and Rueckert, 1988). nutrient absorption and macromolecule production (McGhee, 2007). This is the first report describing the sites of viral protein We detected as many as six discrete intestinal cells that expressed high expression in naturally infected Caenorhabditis nematodes. Many levels of viral protein in infected larval animals. While in some ways taxonomically related viruses have distinct tropisms, so it was unclear it is quite striking that so few cells in an entire animal appeared to be at the outset whether these three viruses would have common or infected, these infected cells represent up to 30% of the total intestinal unique tropisms. For example, in mammalian viruses, members of the tissue. The effects of virus infectiononintestinalcellfunctionin family Picornaviridae can infect liver (hepatitis A), the respiratory tract Caenorhabditis nematodes are currently unknown. The 20 intestinal (rhinoviruses), the enteric tract (enteroviruses), and the central cells arise during development from distinct progenitor cells and are nervous system (enteroviruses) (Whitton et al., 2005). We demon- divided into 9 “ints” based on their origin (McGhee, 2007). Differences strated by IFA that all three viruses shared a common primary tropism in gene expression and anatomy between anterior and posterior of the intestine, despite extensive evolutionary divergence. Le Blanc, intestinal cells have been reported in C. elegans (Schroeder and Santeuil and Orsay viruses share less than 50% amino acid identity in McGhee, 1998). In most cases, the positive staining cells were located the RdRP (Felix et al., 2011; Franz et al., 2012). Defining the tropism of in the anterior portion of the intestine, raising the question of whether these three viruses is a critical step forward in the development of the different intestinal cells may be differentially susceptible to these models for studying virus–host interactions. With the knowl- infection. For example, one possibility is that only a subset of intestinal edge that all three viruses primarily infect the intestine, each of the cells expresses the appropriate receptor for viral entry. Alternatively, viruses can be used as a model to better understand intestinal the observation may simply reflect that these are the first cells to infection and disease. Moreover, the fact that they share a similar encounter the virus as the virus particles transit the intestinal lumen. tropism will enable more robust comparative analysis among infec- In some cases, Orsay virus-infected animals had a greater number tions since they infect the same type of cells. Ultimately, the general of RdRP-positive cells than capsid-positive cells (Figs. 3B–E, 7, 8Aand strategy of using virus infection of nematodes as a model for human C). This observed difference might reflect trivial technical sensitivity disease will be strengthened by the identification of additional viruses issues (i.e. the capsid antibody is less sensitive than the RdRP antibody) with distinct tropisms such as muscle or neurons to complement or biological differences in the expression levels of the capsid and these existing viruses. In the not too distant future, we may have the RdRP. It is possible that the capsid-negative, RdRP-positive cells were ability to compare and contrast the nematode host response to a in an early stage of infection and expressed greater levels of RdRP than variety of viral infections in different tissues. capsid. Another alternative explanation is that those cells may be undergoing a form of abortive infection that leads to either the absence of or low levels of expression of the capsid. Materials and methods For Orsay virus, we observed mutually exclusive subcellular localization of the RdRP and capsid in the cytoplasm of infected cells Strains (Fig. 8). The RdRP and capsid proteins of nodaviruses are known to localize in the cytoplasm of host cells and associate with different host Isolation of wild C. elegans strain JU1580 and wild C. briggsae organelles. For example, FHV RdRP inserts and forms spherules in strains JU1264 and JU1498 has been described (Felix et al., 2011; the outer membranes of host mitochondria (Miller et al., 2001). Franz et al., 2012). N2 and WM27 (rde-1 mutant) were obtained OnepossiblefunctionofthisistosequesterthedsRNAreplicative from the Caenorhabditis Genetics Center (CGC). intermediates away from host cell RNAi machinery. Boolarra nodavirus infection in Drosophila cells is also associated with mitochondria, Infectious filtrate preparation resulting in the loss of mitochondria by 24 hpi (Bashiruddin and Cross, 1987). The FHV capsid is known to associate with the host C. elegans strain JU1580 infected with Orsay virus and C. endoplasmic reticulum (Venter et al., 2009). In addition, in Drosophila briggsae strain JU1264 infected with Santeuil virus or Le Blanc 262 C.J. Franz et al. / Virology 448 (2014) 255–264

Fig. 8. Subcellular localization of Orsay viral proteins in C. elegans using confocal microscopy. Orsay virus-infected JU1580 (A and B) and rde-1 mutant (C) larvae were stained with polyclonal goat anti-RdRP and polyclonal rabbit anti-capsid antibodies. White boxes depict enlarged regions of interest in the panels directly below. a: capsid staining at the apical surface of intestinal cells, p: punctate capsid staining patterns in the cytoplasm, n: nucleus, and v: vesicle-like. Scale bars represent 20 μm. virus were grown at 20 1C on 60 Nematode Growth Medium Supernatant was removed and the pellet (3 ml) was homoge- (NGM) plates (10 cm) seeded with 1 ml OP50 bacteria. When nized using a MagNA Lyser Instrument (Roche) in 3 separate 1 ml animals reached starvation, each plate was rinsed with 5 ml of aliquots. After centrifugation, the supernatant was removed then M9 and centrifuged at 3000 RPM for 2 min to harvest animals. ﬁltered through a 0.22 mm pore-sized membrane (Millipore). M9 Plates were rinsed again to collect the remaining animals. buffer was added to each aliquot to reach a ﬁnal volume of 5 ml. C.J. Franz et al. / Virology 448 (2014) 255–264 263

The Orsay virus titer (RNA copies/ml) was determined using a Orsay RdRP antibodies. To determine the percentage of larvae that TaqMan real-time RT-PCR and comparison to a standard curve. were infected by virus at each time point, 100 animals per time Primers and probe used for this assay were 5′ CCGGCGACAATGTG- point were counted and the number of animals with detectable TACCA (GW303), 5′ CCAGCCCTCCGTTGACAA (GW304), and 5′ FAM/ levels of viral protein was determined. In each of the positive CGAGGCCACTATCAGGG. The resulting amplicon was 68 bp. A animals, it was noted whether the animal was positive for RdRP, standard curve was generated by making serial 10-fold dilutions capsid or both proteins. Three independent experiments were of an in vitro transcribed Orsay RNA2 fragment ranging from performed and the mean and standard deviation were calculated. 5 107 to 5 copies per reaction. PCR mixtures consisted of 2 universal TaqMan real-time RT-PCR master mix (Applied Biosys- Fluorescence in situ hybridization assay (FISH) tems), 12.5 pmol of each primer, 1 ul of 25 probe, 0.625 ul of 40 RNAse inhibitor (Applied Biosystems), and 5 ul of sample. An aliquot of the infected animals from the time course Orsay infected samples were run as 1:1000 dilutions of the experiment (see above) was set aside for FISH, which was original sample. The cycling conditions (BioRad CFX96) were: performed as previously described (Raj et al., 2008) using custom 48 1C for 30 min, 95 1C for 15 min, and 40 cycles of 95 1C for Stellaris™ (Biosearch Technologies) probes labeled with Quasars 15 s, 60 1C for 1 min. 670 Dye for the Orsay virus RNA1 molecule and probes labeled with Cal Fluor Reds 610 Dye for the Orsay virus RNA2 molecule. Immunofluorescence assay The oligonucleotide sequences used for the probes are given in Supplemental Table S1. Standard fluorescence microscopy was Polyclonal peptide antibodies were produced by Genscript using performed using an upright Zeiss AxioImager M1 equipped with the following peptide sequences: rabbit and goat anti-Orsay RdRP 63 , 1.25 numerical aperture and 100 , 1.3 numerical aperture (GDRRDDPHAPYKAT); rabbit and goat anti-Orsay capsid (APTRGA- objectives. Images were acquired using a Pixis 1024B camera DAVREYCP); rabbit anti-Santeuil RdRP (CCADVSKMDA); rabbit anti-Le (Princeton instruments) and MetaVue™ imaging software pro- Blanc RdRP (CNQRRWAARGRPRGR). grammed to acquire Z sections spaced 0.7 mm apart. Image panels The following samples were prepared: Orsay virus-infected and were assembled in Adobe Photoshop CS. The number of intestinal mock-infected (M9 buffer) rde-1 mutant, JU1580 and N2; Santeuil cells showing intracellular staining after exposure times ranging virus-infected and mock-infected JU1264; Le Blanc virus-infected from 50 to 3000 ms was quantified. One cell was considered and mock-infected JU1498. For all experiments, animals were positive for infection when the signal, for exposure less than or maintained at 20 1C on NGM plates, then fixed for 10 min in equal to 3000 ms, was distinct from the non-specific background Bouin's fixative (0.75 ml of saturated picric acid, 0.25 ml of staining observed on mock-infected controls. formalin, 0.05 ml of glacial acetic acid, 0.25 ml of methanol, and 0.01 ml of β-mercaptoethanol) (Nonet et al., 1997). Fixed animals were incubated in blocking solution (1 PBS, 0.5% BSA, 0.5% Acknowledgments Triton X-100, and 10 mM sodium azide) for 2 h. The animals were then incubated with either a single primary antibody or two The N2 and WM27 strains were provided by the CGC, which is primary antibodies overnight at 4 1C. All viral protein antibodies funded by NIH Office of Research Infrastructure Programs (P40 were diluted 1:100 in blocking solution. A 1:250 dilution was used OD010440). The monoclonal antibody MH33 developed by Francis for the MH33 antibody (Developmental Studies Hybridoma Bank) and Waterston was obtained from the Developmental Studies (Francis and Waterston, 1991). Animals were washed multiple Hybridoma Bank developed under the auspices of the NICHD times in blocking solution and then incubated overnight at 4 1C and maintained by The University of Iowa, Department of Biology, with a 1:500 dilution of the appropriate secondary antibody. The Iowa City, IA 52242. DW holds an investigator in the pathogenesis s Alexa Fluor secondary antibodies (Invitrogen) used were donkey of infectious diseases award from the Burroughs-Wellcome Fund. anti-rabbit 488 and 568; donkey anti-goat 488 and 568; goat anti- This work was supported by a grant from the Agence Nationale rabbit 488; goat anti-mouse 546. Nuclei were counterstained for pour la Recherche (ANR 11 BSV3 01301) and a Coup d'Elan de la 15 min with Hoechst (Invitrogen) or 20 min with TO-PRO3 (Invi- Fondation Bettencourt-Schueller to MAF. We thank Michael Nonet trogen) following the manufacturer's protocol. Epifluorescent and members of his laboratory for helpful discussions concerning images were taken with a Zeiss Axioskop Mot Plus fluorescence IFA, and Michalis Barkoulas for great help with FISH. microscope equipped with a 63 , 1.4 numerical aperture Zeiss Plan Apochromat oil objective. Images were acquired using Axio- vision 4.6 software (Carl Zeiss Inc.). Confocal images were acquired Appendix A. Supporting information using a Zeiss LSM510 Meta laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a 63 , 1.4 numer- Supplementary data associated with this article can be found in ical aperture Zeiss Plan Apochromat oil objective. Confocal Z the online version at http://dx.doi.org/10.1016/j.virol.2013.09.024. sections of 0.33 mm were obtained using the Zeiss LSM510 software. Image panels were assembled in Adobe Photoshop CS. References

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