Arch Virol (2014) 159:2843–2851 DOI 10.1007/s00705-014-2168-2
BRIEF REVIEW
The nematode Caenorhabditis elegans as a model to study viruses Jesica Diogo • Ana Bratanich
Received: 28 February 2014 / Accepted: 28 June 2014 / Published online: 8 July 2014 Ó Springer-Verlag Wien 2014
Abstract Caenorhabditis elegans is a worm that has been extensively studied, and it is today an accepted model in many different biological fields. C. elegans is cheap to maintain, it is transparent, allowing easy localization studies, and it develops from egg to adult in around 4 days. Many mutants, available to the scientific community, have been developed. This has facilitated the study of the role of particular genes in many cellular pathways, which are highly conserved when compared with higher eukaryotes. This review describes the advantages of C. elegans as a laboratory model and the known mechanisms utilized by this worm to fight pathogens. In particular, we describe the strong C. elegans RNAi machinery, which plays an important role in the antiviral response. This has been shown in vitro (C. elegans cell cultures) as well as in vivo (RNAi-deficient strains) utilizing recently described viruses that have the worm as a host. Infections with mammalian viruses have also been achieved using chemical treatment. The role of viral genes involved in pathogenesis has been addressed by evaluating the phenotypes of transgenic strains of C. elegans expressing those genes. Very simple approaches such as feeding the worm with bacteria transformed with viral genes have also been utilized. The advantages and limitations of different approaches are discussed.
J. Diogo A. Bratanich (&) Department of Virology, School of Veterinary Sciences, University of Buenos Aires, Av. Chorroarin 280, 1427 Buenos Aires, Argentina e-mail: [email protected]
The C. elegans model In the year 1998, the Caenorhabditis elegans (C. elegans) consortium completed the sequence of the genome of this free-living nematode, which is calculated to contain around 20,470 genes. The name is a mixture of Greek (caeno- – recent, rhabditis – rod-like) and Latin (elegans – elegant). Since the time that Sydney Brenner developed this model, an enormous number of scientific papers have been published validating the use of this nematode as a model to study many cellular processes. A great part of its success is due to the many similarities between nematode and human genes. Many human genetic disorders are based on genes with homologues in C. elegans [1]. In addition, C. elegans has a number of advantages. It is a very economical model that only needs bacteria to survive. When bacteria are not available, C. elegans does not die but instead enters into a state of developmental arrest called dauer, with special morphologic and physiologic characteristics. In three to four days, the entire life cycle is completed with the development and release of eggs from hermaphrodites to initiate a new generation. An adult worm can lay around 300 eggs, which will develop into larvae within hours [2]. The adult hermaphrodite has 959 somatic cells covering different kinds of tissues such as muscle, intestine, germline and neurons. The nervous system of the C. elegans hermaphrodite is composed of 302 neurons grouped in ganglia. Expression of proteins in specific tissues can be achieved with the use of selected promoters [3]. Due to their sexual dimorphism (hermaphrodite and male), C. elegans is a good model for genetic studies. The possibility of autofecundation in the hermaphrodite is useful to maintain homozygosis of mutations without the necessity of mating [4].
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In addition, the worm body is transparent, making it easy to observe cellular events or to use fluorescent markers to study in vivo processes such as cell localization or tissue-specific expression [5]. Many mutant strains of C. elegans have been obtained and are available to researchers from the CGC (Caenorhabditis Genetics Center). Mutants are important as a tool to study phenotypic as well as behavioral changes associated with particular genes [6]. C. elegans strains resistant or hypersensitive to particular pathogens have been easily obtained through chemical mutagenesis [7, 8]. Many of these mutations are not lethal in C. elegans. For example, while programmed cell death is a conserved physiological process, it is not essential for survival [9]. Surprisingly, RNAi (RNA interference) experiments are very easy to perform in C. elegans, and it has been successfully used to analyze the functions of many genes [2, 10]. Basically, the procedure consists in stimulating bacteria transformed with a plasmid construct containing the RNAi, which is later given as food to the worm. As explained below, the RNAi effect in C. elegans can be observed in all tissues except neurons and can even be transmitted to its progeny [11]. In addition, unlike higher eukaryotes such as mammals, the RNAi response can be amplified using a specific enzymatic machinery [12]. In the laboratory, transgenic strains can be obtained by injecting a plasmid carrying the gene of interest into the gonads of the animal. This microinjection is done together with a plasmid carrying a marker gene (such as GFP or phenotypic dominant markers) to facilitate the identification of transgenic animals. Injected plasmids remain as extrachromosomal arrays, and the transgenes are expressed according to the promoter used. If stable expression is desired, gamma irradiation can be used to incorporate the sequences into the genome of the worm, but this procedure requires the stocks to be purified through crosses with wildtype animals to eliminate undesired mutations. Another approach to obtain stable strains is gene bombardment [13]. C. elegans strains can be stored frozen for long periods of time.
C. elegans response against microbial and viral pathogens C. elegans has been utilized as a model in studies related to microbial pathogenesis and innate immunity only in recent years when it was observed that the worm was susceptible to various human (as well as natural) pathogens such as bacteria and fungi [14]. As a consequence, C. elegans genes that are important for its defense mechanism have been identified and characterized since then. Many of these genes have been found to share strong homology with
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similar genes in other species, suggesting the existence of a common evolutionary origin [15]. C. elegans lacks an adaptive immune system and only uses an innate response system to fight pathogens [16]. This response is based on avoidance behaviors and physical barriers. When C. elegans is exposed to a pathogenic microorganism, protective mechanisms are activated, including a repulsive behavior provoked by the detection of specific microbial molecules [17, 18]. Apparently, C. elegans does not possess homologues of many of the major pattern-recognition modules present in other organisms. Although some homologues of the Toll pathway are present in the worm (such as tol-1, trf-1, pik-1, and ikb-1), none of them seem to be essential for resistance. It has been observed that tol-1 mutants show less avoidance of certain pathogens, such as Serratia marcescens and Salmonella [17, 19]. Tol-1 is the only TLR (Toll-like receptor) encoded in C. elegans. When the pathogen cannot be evaded, C. elegans triggers an innate immune response, which leads to the activation of specific signaling pathways than in turn produce and release defense molecules [15, 20]. There are at least four signaling pathways that modulate the C. elegans response to pathogens: ERK kinase, MAP kinase, TGF-b (DLB-1 pathway) and the insulin-DAF-2/DAF16 circuit [21, 22]. Among the immune effectors produced, there are a wide variety of antimicrobial peptides (AMPs) and proteins such as lysozymes and lectins that lead to a systemic immune resistance state [15, 16]. Unlike microbes, the C. elegans defense mechanisms against viruses are based on the RNAi pathway, which is highly conserved in plants and animals.
The RNAi antiviral machinery The utilization of C. elegans to study viral pathogenesis and antiviral innate immunity was delayed compared to its use for bacterial or fungal infections. This was due in part to the fact that the worm is not easily infected by mammalian viruses, but also because viruses infecting and replicating naturally in this model were not discovered until recently (Table 1). As a consequence, it has been difficult to identify the mechanisms of defense against this kind of pathogen. However, it was quickly found that C. elegans shared components of the RNAi pathway with higher eukaryotes [23, 24]. The C. elegans RNAi machinery is composed of a single Dicer enzyme (DCR-1), four RNA-dependent RNA polymerases (RDRs)(ego-1 and rrf-1–rrf-3) and 27 Argonaute (AGO) proteins, which play a role in different RNA pathways [25–27]. This RNAi machinery differs from that of mammals, in which there are two kinds of RNAse III ribonucleases, Drosha and Dicer, participating in the
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Table 1 Summary of established viral models for Caenorhabditis elegans Type of strategy
Virusa family
Genome
Experimental features
References
In vitro infection
VSV Rhabdoviridae
ssRNA(-)
[37]
In vivo infection
VV Poxviridae
dsDNA covalently joined ends
Natural and experimental infection
OrV Nodaviridae
ssRNA(?) bipartite
Transgenic expressing a fragment of a viral genome
FHV Nodaviridae
ssRNA(?) bipartite
Transgenic expressing a viral gen
AcMNPV Baculoviridae
dsDNA circular
HDV Unassigned
ssRNA(-)
HBV Hepadnaviridae
dsDNA circular (gapped)
HIV-1 Retroviridae
ssRNA(?) (2 copies)
VSV engineered to encode a GFP fusion protein was utilized to infect wild-type and RNAi-defective cells of C. elegans. Cells lacking components of the RNAi apparatus produced more GFP and infective particles than wild-type cells. Furthermore, mutant cells with enhanced RNAi produced less GFP. A recombinant VV expressing a lacZ reporter gene was used to monitor viral genome replication. The VV entry was facilitated by a PEG-mediated infection protocol. Blue X-Gal staining was observed in many tissues and organs. Virus replication was significantly enhanced in ced-3, ced-4, ced-9(gf), and egl-1(lf) mutants, demonstrating that the core programmed cell death genes control VV replication in C. elegans. OrV persisted in C. elegans cultures through horizontal transmission, causing major damage in intestinal cells, albeit with remarkably little effect on the animal; worms continued moving, eating, and producing progeny, although at a lower rate. C. elegans rde-1 mutation (RNAi pathway) conferred susceptibility to the OrV. Worm strains were generated to carry either a chromosomallyintegrated FHV RNA1 or RNA2 transgene. Drosophila culture cells were transfected with RNA extracted from worms to verify the biological activities of FHV RNAs. FHV replication triggered potent antiviral silencing in C. elegans that requires RDE-1. The worm immunity was capable of rapid virus clearance in the absence of expression of FHV B2 protein, a RNAi inhibitor. The p35 gene is an inhibitor of virus-induced apoptosis in insect cells. The expression of p35 in C. elegans prevented death of cells normally programmed to die. This suppression of developmentally programmed cell death resulted in the appearance of extra surviving cells. Expression of p35 could prevent the embryonic lethality of a mutation in ced-9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl-2. HDAg is a multifunctional protein involved in pathogenesis. Ubiquitous expression of HDAg in transgenic worms resulted in 20 % to 70 % sterility, while worms expressing HDAg in the pharynx displayed 70 % sterility. Most worms expressing HDAg in pharynx were arrested at larval stage 2 or 3 and displayed a 70 % reduction in brood size. HBx is a multifunctional protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. The expression of HBx in C. elegans induced apoptosis and necrosis, which mimics one of the early cellular events following liver infection by HBV. HBx-induced cell death pathways involved cell death regulators and executors and components of the necrosis pathway. The Nef protein regulates multiple functions in the host that enhance HIV-1 pathogenesis. The feeding of worms on bacteria transformed with a sequence of Nef resulted in its absorption via the gut. Nef induced several altered physiological effects in animals such as lipodystrophy, impaired locomotion, compromised reproductive performance and reduced life span. These physiological effects were comparable to with pathologic symptoms in mammals.
Feeding worms with a viral protein
[46]
[34, 48, 54]
[38]
[49]
[50]
[51]
[52]
a Abbreviations: AcMNPV, Autographa californica multiple nucleopolyhedrovirus; FHV, Flock House virus; HBV, hepatitis B virus; HDV, hepatitis D virus; HIV-1, human immunodeficiency virus 1; OrV, Orsay virus; VSV, vesicular stomatitis virus; VV, vaccinia virus
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biogenesis of miRNAs and siRNAs, respectively [28]. Unlike insects and plants, which dedicate the entire RNAi machinery to the antiviral response, worms use it for the regulation of physiological processes as well [29]. For example, mutations in ego-1 disrupt RNAi for some germline genes and lead to defects in germline development, and animals with a defective dcr-1 gene are sterile [25, 30]. In these physiological events, DCR-1 processes stem-loop structures that are characteristic of precursor microRNAs (pre-miRNAs) to generate mature microRNAs (miRNAs), apparently without a requirement for other factors [31]. DCR-1 also acts on both exogenous (dsRNA microinjection or feeding) and endogenous double-stranded RNAs (aberrant endogenous dsRNA) (Fig. 1). For exogenous dsRNA, the process requires, aside from DCR-1, the binding of the RNA-binding protein RDE-4 [32]. During viral infection, similar components are recruited, but the process is initiated by an RIG-1 homologue called DHR-1. DHR-1 recruits DCR-1 and its partner RDE-4 to the viral dsRNA replication intermediate. The processing by DCR-1 generates primary siRNAs that then bind to the Argonaute protein RDE-1. Mutation of Rde-1 leads to higher viral loads, confirming the role of the RNAi system as an antiviral mechanism [33, 34]. After binding to RDE-1, the primary siRNAs can be amplified by RNA-dependent RNA Fig. 1 Description of the main components of the C. elegans RNAi machinery and differences compared to other exogenous sources of dsRNA
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polymerases to generate antisense secondary siRNAs [12]. This process is mediated by primary siRNAs that prime the synthesis of dsRNA using the target mRNA itself as a template. Thus, secondary siRNAs are encoded by the target gene but not by the trigger RNA that originated the primary siRNAs. This amplification process, which generates a systemic effect, does not occur in mammals [12]. Secondary siRNAs target the viral genome and are thus called viRNAs [35]. This targeting of the viral RNA by viRNAs is the signal for the destruction of the molecule by specific AGO proteins (SAGO-2) [36].
Strategies to study viruses in C. elegans Infection of C. elegans cells in vitro In this approach, normal and RNAi-defective primary embryonic C. elegans cells were infected with a recombinant vesicular stomatitis virus (VSV, family Rhabdoviridae), Indiana strain, carrying a GFP-P gene fusion protein to facilitate evaluation (Table 1) [37]. The embryonic cell culture was produced from eggs purified from bleached gravid worms (bleach is used to eliminate contaminant bacteria and to synchronize worm cultures). Cells obtained from eggs after enzymatic treatment were seeded at
The nematode Caenorhabditis elegans
3 9 106 cells per cm2 in eight-well chambered coverglasses coated with peanut lectin and maintained at 27 °C in a sealed humid chamber until infection. Twenty to twenty-four hours after exposure to virus at different moi, infection was detected through the presence of green fluorescent cells; infectious virus from infected worm cells was evaluated by plaque assays on Vero cells, and the relative concentrations of VSV negative-strand RNA were measured by quantitative PCR of cDNA. To evaluate the ability of the VSV virus to spread among cells, a neutralizing antibody targeting the viral attachment protein was used to treat infected cells. Using this approach, a reduction of the number of infected cells was observed, suggesting that the virus was able to spread from cell to cell. After seven days of infection, cells with a defective RNAi apparatus (mutations in rde-1 and rde-4 genes) produced more GFP, genomic minus RNA, and infectious particles than uninfected cells. When using C. elegans strains with enhanced RNAi machinery (like, for example, cells defective in nucleases that normally degrade siRNAs), the result was the opposite. These experiments showed that resistance to VSV infection correlated with a normal RNAi machinery, demonstrating the importance of this mechanism in the antiviral response.
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In this approach, segments of Flock House virus (FHV) (family Nodaviridae), which has a bipartite positive-sense RNA genome, were introduced into C. elegans by microinjection [38] (Table 1, Fig. 2). When a DNA fragment is microinjected into worm gonads, it is maintained in extrachromosomal arrays in the generated progeny and is expressed according to the promoter that is selected.
FHV was chosen in this work because it could replicate in different types of cells (plant, yeast, insect and mammalian) [39]. Segment 1 (RNA1) encodes the viral RNAdependent RNA polymerase, and it can replicate independently of segment 2 (RNA2), which encodes the viral capsid protein precursor. At its 30 end, FHV RNA1 encodes a subgenomic RNA3 containing two overlapping open reading frames (ORFs) encoding proteins B1 and B2. B2 is an RNAi suppressor that is active across the animal and plant kingdoms [40]. Transgenic strains of C. elegans carrying these segments under the control of a heat-inducible promoter generated all of the transcripts produced during normal viral replication. The detection of self-replication of RNA1 and transreplication of RNA2 together with the production of subgenomic RNA was taken as evidence of viral replication, although no viral particles were shown to be present. In addition, it was observed that the replication of the virus was inhibited via an RNAi mechanism mediated by the B2 protein. When the B2 gene was mutated, viral replication increased [38]. In a later study, the FHV system was utilized to identify genes that are essential for the antiviral response, such as the previously mentioned RDE-1 protein, which was found to be essential for siRNAs but not for miRNAs [26]. In turn, the broad-spectrum RNAi inhibitor FHV B2 protein was found to act upstream of rde-1 by targeting the virusderived primary siRNAs and facilitating infection of C. elegans by, for example, Orsay virus [41] (see below). In spite of these findings, this approach does not allow the host response to the whole virus to be studied, since a real infection is not happening. As a consequence, some details of the replication cycle and the ability of the virus to propagate in other hosts cannot be studied.
Fig. 2 Genomic characteristics of the Flock House virus (FHV) genome. Segment 1 (RNA1) encodes the RNA-dependent RNA polymerase; segment 2 (RNA2) encodes the capsid protein. The B2
protein is produced from a subgenomic RNA3. RNA1 and RNA 2 were cloned downstream of a heat-inducible promoter and microinjected into C. elegans gonads to obtain transgenic strains
Transgenic C. elegans expressing a fragment of a viral genome
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In vivo infection of C. elegans with mammalian viruses
Natural infection of C. elegans by viruses
By utilizing polyethylenglycol (PEG) to make the cuticle permeable, C. elegans larva (L3/L4 stages) could be infected with a recombinant vaccinia virus (VV, family Poxviridae) expressing b-galactosidase/CAT (chloramphenicol acetyltransferase) and with a recombinant Sindbis virus expressing GFP (green fluorescent protein). PEG was used to facilitate viral entry, since natural infection with these viruses could not be achieved (Table 1). The CAT gene was cloned under the control of an early VV promoter, whereas the b-galactosidase gene was cloned under the control of a late one; both genes were only turned on if the recombinant virus was undergoing productive genome replication. Thus, the level of expression of X-gal and bgalactosidase was taken as a measure of the level of viral replication. This was also analyzed by real-time PCR and western blotting. Parameters such as viral titers, PEG size and concentration, incubation time and temperature, and the developmental stage of the worms were tested. It was found that 5 % of early adults could be infected with 1 9 107 PFU /ml in the presence of 2 % PEG 1000 for 5 minutes at room temperature. The percentage could be increased to 18 % by moving the worms to a buffer (M9) with the same viral concentration for 6 hours at room temperature. Viral particles were observed by electron microscopy, and viable virus could be recovered from several worm tissues. Blue X-Gal staining was observed in many tissues and organs, including pharynx, intestine, muscle, hypodermis, and occasionally germline. Parameters of infection could only be detected for 3 days postinfection (p.i.), suggesting that some restriction to VV replication existed in the wt worm. By day 4, 80 % of the infected worms had died, whereas there were no dead animals when infection was performed with UV-treated virus. Next, the authors investigated the role of programmed cell death in the observed restriction of viral infection. In C. elegans, four genes control programmed cell death: ced3, ced-4, ced-9 and egl-1. The first three encode proteins similar to human caspases, Apaf-1 and Bcl-2, respectively [42–44], whereas EGL-1 is a small BH3-containing protein [45]. Mutations in all of these genes block most of the programmed cell death, and fewer apoptotic bodies can then be seen during the development of the worm. This is quite evident in the pharynx, where more than 80 % of the cells that normally die survive. By means of different C. elegans strains mutated in this apoptotic pathway, the authors found a role for caspases inhibiting infection [46]. All of the findings described for VV infection were also observed with the recombinant Sindbis virus expressing GFP.
Very recently, two new RNA viruses with similarities to members of the family Nodaviridae were described in natural populations of Caernorhabditis worms. One of them (Orsay virus) infected C. elegans naturally, whereas Santeuil virus could do the same in C. briggsae, another worm species from the same genus as C. elegans (Table 1) [34]. Both of them were identified by a pyrosequencing approach from animals with abnormal morphologies in intestinal cells (storage granules disappeared and cytoplasm became fluid) in which small virus-like particles of approximately 20 nm diameter were visible by electron microscopy. As described previously, viruses in this family have a bipartite genome, with segment one (RNA1) encoding the RNA-dependent RNA polymerase and segment two (RNA2) encoding the capsid protein. Some members of this family, as shown for FHV, encode proteins B1 and B2 from a subgenomic RNA produced from RNA1. The putative RNA-dependent RNA polymerase and capsid proteins of Orsay virus shared around 30 % amino acid sequence identity with similar genes of other nodaviruses, whereas the identity between the Orsay and Santuiel virus RNA-dependent RNA polymerases was only of 44 %. Interestingly, both viruses were predicted to encode a second ORF (ORF d) in the 30 half of the RNA2 segment in addition to the capsid protein at the 50 end of the same fragment. Also, neither virus appeared to encode a B1 or a B2 protein. These features suggested that the genomic organization of these viruses was distant from that of other members of the same family. Furthermore, phylogenetic analysis of the predicted RNA polymerase and capsid proteins of both viruses showed that they were highly divergent from those of other nodaviruses, suggesting that these viruses might represent a novel genus. In vivo studies with these viruses showed that they were not integrated into the nematode genome and that they could not be transmitted vertically in spite of being present in worm cultures for longer than 50 generations (6 months). These results suggested that horizontal transmission of the virus occurs, and that these nematodes are a suitable model for the study of a virus in its natural host. In addition, C. elegans mutants deficient in the RNAi machinery (red-1, rde-4) could be infected more easily than the wild type (N2 Bristol strain), suggesting again an antiviral role of the RNAi response. These mutants showed higher levels of virus replication and more symptoms [34]. Furthermore, it was observed that natural isolates of C. elegans showed distinctive susceptibility to infection with Orsay virus, a sign of natural variation in host antiviral defenses.
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Recently, by high-throughput sequencing of wild Caenorhabditis strains, a new virus was identified in a C. briggsae isolate. This virus, called Le Blanc, was found to be distantly related to Orsay virus and Santeuil virus but still showed an overall similarity to nodaviruses [47]. More recently, studying the whole transcriptome in response to infection with Orsay virus, a significant number of virus-specific immune response genes were identified in N2 and rde-1 worms [48]. In particular, the RNAi response to the viral infection was found to be mediated by a redirection of the Argonaute protein RDE-1, leading to loss of repression of its targets. Thus, the authors suggested that the antiviral RNAi response was regulated by a competition between exogenous and endogenous RNAi pathways. Suppressors of the C. elegans RNAi response have recently been identified using the Orsay virus infection approach. In this way, the role of the FHV B2 protein as a broad-spectrum RNAi inhibitor was confirmed. Furthermore, it was found that the B2 protein acted upstream of rde-1 by targeting the virus-derived primary siRNAs and thus facilitating infection of C. elegans by Orsay virus [41]. C. elegans for the study of viral genes One of the first and few published works using C. elegans to evaluate viral genes involved the study of a baculovirus antiapoptotic gene (p35) (Table 1). When worms expressing this gene under a heat shock protein promoter were exposed to heat, there was a reduction in programmed cell death during development. This effect was observed in the embryo as a decreased number of apoptotic bodies and in the adult, as described before, in the form of extra cells in the pharynx (Table 1) [49]. In a more recent work, C. elegans was used to express a multifunctional protein from hepatitis delta virus (HDAg) [50]. HDAg is involved in the pathogenesis of this viral infection, contributing by still unknown mechanisms, to the carcinogenic process developed in hepatocytes. Typical C. elegans characteristics such as sterility, growth, and brood size were evaluated in transgenic strains expressing HDAg under the control of two different promoters, a ubiquitous promoter (fib-1) and a muscle-specific promoter (myo-2) that is only active in the pharynx. Ubiquitous expression of HDAg resulted in 20 to 70 % sterility, whereas transgenic worms expressing the protein in pharynx tissue were arrested at larval stage 2 or 3, with a reduction in brood size. Growth retardation was suggested to be explainable by possible changes in two pathways, TGF-beta and insulin-like. Transgenic C. elegans strains expressing the HBx protein of hepatitis B virus also showed a reduction in apoptotic as well as necrotic processes, similar to what is
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observed in the early stages of liver infection by this virus (Table 1) [51]. In an interesting approach, C. elegans was fed with bacteria expressing the human immunodeficiency virus Nef protein, which is known to be involved in pathogenesis by enhancing viral replication (Table 1). A phenotype with features similar to those of the in vivo infection was obtained. For example, marked lipodystrophy, neuromuscular dysfunction, impaired fertility, and reduced longevity were observed in these worms [52].
Concluding remarks This review attempts to summarize the first experiences in the use of C. elegans as a model to study viral pathogenesis. Firstly, C. elegans is a cheap model that requires minimal equipment and reagents and is only fed with bacteria. Phenotypes can be evaluated very quickly, since the worm becomes adult in 3-4 days. The similarities between C. elegans and higher eukaryotic pathways together with the availability of many mutants in those pathways represent one of its major benefits. In fact, infection by different kinds of bacterial pathogens has been analyzed in this way, allowing the identification of several genes involved in virulence and host defense. On the other hand, unlike bacteria and fungi, the worm cannot be infected very easily with foreign viruses. The diversity of the above-mentioned strategies reflects efforts to establish C. elegans as a model for infection. An artificial infection method has been based on introducing a partial virus genome of Flock House virus into animals [38, 53]. This experimental system can only examine replication of the viral RNA and is fundamentally unable to address the host response to other critical aspects of the virus life cycle such as a virus entry, virion assembly or egress [53]. As mentioned in this review, the only successful artificial infection utilized a chemical treatment (PEG) to infect C.elegans with vaccinia virus [46]. However, no other papers have been published using this approach, or other new ones, suggesting that the worm cuticula may represent a major barrier to these studies. Certainly, the PEG approach makes the study of particular aspects of the viral replicative cycle such as receptor recognition and entry very difficult. Another level of difficulty is added if, for example, transgenic strains have to be produced for expression of particular viral genes. This approach requires expertise in microinjection and a good knowledge of worm genetics. If it can be achieved, however, the effect of many viral genes can be analyzed in the background of different mutations involving highly conserved pathways. Furthermore, because C. elegans is transparent, the progression of
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infection with a marker virus or the expression of a particular viral gene in a specific tissue can be easily monitored. The discovery of nodaviruses that naturally infect C. elegans and its close relative C. briggsae, has generated new expectations. This virus has shown the ability to be transmitted horizontally, causing major damage in intestinal cells [46, 53]. Nodavirus infections are therefore becoming a feasible system for studies on viral-host interaction, specificity, immune response, pathogenesis and evolution. The finding of natural infections in C. elegans will certainly provide a deeper comprehension of the viral life cycle.
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BRIEF REVIEW
The nematode Caenorhabditis elegans as a model to study viruses Jesica Diogo • Ana Bratanich
Received: 28 February 2014 / Accepted: 28 June 2014 / Published online: 8 July 2014 Ó Springer-Verlag Wien 2014
Abstract Caenorhabditis elegans is a worm that has been extensively studied, and it is today an accepted model in many different biological fields. C. elegans is cheap to maintain, it is transparent, allowing easy localization studies, and it develops from egg to adult in around 4 days. Many mutants, available to the scientific community, have been developed. This has facilitated the study of the role of particular genes in many cellular pathways, which are highly conserved when compared with higher eukaryotes. This review describes the advantages of C. elegans as a laboratory model and the known mechanisms utilized by this worm to fight pathogens. In particular, we describe the strong C. elegans RNAi machinery, which plays an important role in the antiviral response. This has been shown in vitro (C. elegans cell cultures) as well as in vivo (RNAi-deficient strains) utilizing recently described viruses that have the worm as a host. Infections with mammalian viruses have also been achieved using chemical treatment. The role of viral genes involved in pathogenesis has been addressed by evaluating the phenotypes of transgenic strains of C. elegans expressing those genes. Very simple approaches such as feeding the worm with bacteria transformed with viral genes have also been utilized. The advantages and limitations of different approaches are discussed.
J. Diogo A. Bratanich (&) Department of Virology, School of Veterinary Sciences, University of Buenos Aires, Av. Chorroarin 280, 1427 Buenos Aires, Argentina e-mail: [email protected]
The C. elegans model In the year 1998, the Caenorhabditis elegans (C. elegans) consortium completed the sequence of the genome of this free-living nematode, which is calculated to contain around 20,470 genes. The name is a mixture of Greek (caeno- – recent, rhabditis – rod-like) and Latin (elegans – elegant). Since the time that Sydney Brenner developed this model, an enormous number of scientific papers have been published validating the use of this nematode as a model to study many cellular processes. A great part of its success is due to the many similarities between nematode and human genes. Many human genetic disorders are based on genes with homologues in C. elegans [1]. In addition, C. elegans has a number of advantages. It is a very economical model that only needs bacteria to survive. When bacteria are not available, C. elegans does not die but instead enters into a state of developmental arrest called dauer, with special morphologic and physiologic characteristics. In three to four days, the entire life cycle is completed with the development and release of eggs from hermaphrodites to initiate a new generation. An adult worm can lay around 300 eggs, which will develop into larvae within hours [2]. The adult hermaphrodite has 959 somatic cells covering different kinds of tissues such as muscle, intestine, germline and neurons. The nervous system of the C. elegans hermaphrodite is composed of 302 neurons grouped in ganglia. Expression of proteins in specific tissues can be achieved with the use of selected promoters [3]. Due to their sexual dimorphism (hermaphrodite and male), C. elegans is a good model for genetic studies. The possibility of autofecundation in the hermaphrodite is useful to maintain homozygosis of mutations without the necessity of mating [4].
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In addition, the worm body is transparent, making it easy to observe cellular events or to use fluorescent markers to study in vivo processes such as cell localization or tissue-specific expression [5]. Many mutant strains of C. elegans have been obtained and are available to researchers from the CGC (Caenorhabditis Genetics Center). Mutants are important as a tool to study phenotypic as well as behavioral changes associated with particular genes [6]. C. elegans strains resistant or hypersensitive to particular pathogens have been easily obtained through chemical mutagenesis [7, 8]. Many of these mutations are not lethal in C. elegans. For example, while programmed cell death is a conserved physiological process, it is not essential for survival [9]. Surprisingly, RNAi (RNA interference) experiments are very easy to perform in C. elegans, and it has been successfully used to analyze the functions of many genes [2, 10]. Basically, the procedure consists in stimulating bacteria transformed with a plasmid construct containing the RNAi, which is later given as food to the worm. As explained below, the RNAi effect in C. elegans can be observed in all tissues except neurons and can even be transmitted to its progeny [11]. In addition, unlike higher eukaryotes such as mammals, the RNAi response can be amplified using a specific enzymatic machinery [12]. In the laboratory, transgenic strains can be obtained by injecting a plasmid carrying the gene of interest into the gonads of the animal. This microinjection is done together with a plasmid carrying a marker gene (such as GFP or phenotypic dominant markers) to facilitate the identification of transgenic animals. Injected plasmids remain as extrachromosomal arrays, and the transgenes are expressed according to the promoter used. If stable expression is desired, gamma irradiation can be used to incorporate the sequences into the genome of the worm, but this procedure requires the stocks to be purified through crosses with wildtype animals to eliminate undesired mutations. Another approach to obtain stable strains is gene bombardment [13]. C. elegans strains can be stored frozen for long periods of time.
C. elegans response against microbial and viral pathogens C. elegans has been utilized as a model in studies related to microbial pathogenesis and innate immunity only in recent years when it was observed that the worm was susceptible to various human (as well as natural) pathogens such as bacteria and fungi [14]. As a consequence, C. elegans genes that are important for its defense mechanism have been identified and characterized since then. Many of these genes have been found to share strong homology with
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similar genes in other species, suggesting the existence of a common evolutionary origin [15]. C. elegans lacks an adaptive immune system and only uses an innate response system to fight pathogens [16]. This response is based on avoidance behaviors and physical barriers. When C. elegans is exposed to a pathogenic microorganism, protective mechanisms are activated, including a repulsive behavior provoked by the detection of specific microbial molecules [17, 18]. Apparently, C. elegans does not possess homologues of many of the major pattern-recognition modules present in other organisms. Although some homologues of the Toll pathway are present in the worm (such as tol-1, trf-1, pik-1, and ikb-1), none of them seem to be essential for resistance. It has been observed that tol-1 mutants show less avoidance of certain pathogens, such as Serratia marcescens and Salmonella [17, 19]. Tol-1 is the only TLR (Toll-like receptor) encoded in C. elegans. When the pathogen cannot be evaded, C. elegans triggers an innate immune response, which leads to the activation of specific signaling pathways than in turn produce and release defense molecules [15, 20]. There are at least four signaling pathways that modulate the C. elegans response to pathogens: ERK kinase, MAP kinase, TGF-b (DLB-1 pathway) and the insulin-DAF-2/DAF16 circuit [21, 22]. Among the immune effectors produced, there are a wide variety of antimicrobial peptides (AMPs) and proteins such as lysozymes and lectins that lead to a systemic immune resistance state [15, 16]. Unlike microbes, the C. elegans defense mechanisms against viruses are based on the RNAi pathway, which is highly conserved in plants and animals.
The RNAi antiviral machinery The utilization of C. elegans to study viral pathogenesis and antiviral innate immunity was delayed compared to its use for bacterial or fungal infections. This was due in part to the fact that the worm is not easily infected by mammalian viruses, but also because viruses infecting and replicating naturally in this model were not discovered until recently (Table 1). As a consequence, it has been difficult to identify the mechanisms of defense against this kind of pathogen. However, it was quickly found that C. elegans shared components of the RNAi pathway with higher eukaryotes [23, 24]. The C. elegans RNAi machinery is composed of a single Dicer enzyme (DCR-1), four RNA-dependent RNA polymerases (RDRs)(ego-1 and rrf-1–rrf-3) and 27 Argonaute (AGO) proteins, which play a role in different RNA pathways [25–27]. This RNAi machinery differs from that of mammals, in which there are two kinds of RNAse III ribonucleases, Drosha and Dicer, participating in the
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Table 1 Summary of established viral models for Caenorhabditis elegans Type of strategy
Virusa family
Genome
Experimental features
References
In vitro infection
VSV Rhabdoviridae
ssRNA(-)
[37]
In vivo infection
VV Poxviridae
dsDNA covalently joined ends
Natural and experimental infection
OrV Nodaviridae
ssRNA(?) bipartite
Transgenic expressing a fragment of a viral genome
FHV Nodaviridae
ssRNA(?) bipartite
Transgenic expressing a viral gen
AcMNPV Baculoviridae
dsDNA circular
HDV Unassigned
ssRNA(-)
HBV Hepadnaviridae
dsDNA circular (gapped)
HIV-1 Retroviridae
ssRNA(?) (2 copies)
VSV engineered to encode a GFP fusion protein was utilized to infect wild-type and RNAi-defective cells of C. elegans. Cells lacking components of the RNAi apparatus produced more GFP and infective particles than wild-type cells. Furthermore, mutant cells with enhanced RNAi produced less GFP. A recombinant VV expressing a lacZ reporter gene was used to monitor viral genome replication. The VV entry was facilitated by a PEG-mediated infection protocol. Blue X-Gal staining was observed in many tissues and organs. Virus replication was significantly enhanced in ced-3, ced-4, ced-9(gf), and egl-1(lf) mutants, demonstrating that the core programmed cell death genes control VV replication in C. elegans. OrV persisted in C. elegans cultures through horizontal transmission, causing major damage in intestinal cells, albeit with remarkably little effect on the animal; worms continued moving, eating, and producing progeny, although at a lower rate. C. elegans rde-1 mutation (RNAi pathway) conferred susceptibility to the OrV. Worm strains were generated to carry either a chromosomallyintegrated FHV RNA1 or RNA2 transgene. Drosophila culture cells were transfected with RNA extracted from worms to verify the biological activities of FHV RNAs. FHV replication triggered potent antiviral silencing in C. elegans that requires RDE-1. The worm immunity was capable of rapid virus clearance in the absence of expression of FHV B2 protein, a RNAi inhibitor. The p35 gene is an inhibitor of virus-induced apoptosis in insect cells. The expression of p35 in C. elegans prevented death of cells normally programmed to die. This suppression of developmentally programmed cell death resulted in the appearance of extra surviving cells. Expression of p35 could prevent the embryonic lethality of a mutation in ced-9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl-2. HDAg is a multifunctional protein involved in pathogenesis. Ubiquitous expression of HDAg in transgenic worms resulted in 20 % to 70 % sterility, while worms expressing HDAg in the pharynx displayed 70 % sterility. Most worms expressing HDAg in pharynx were arrested at larval stage 2 or 3 and displayed a 70 % reduction in brood size. HBx is a multifunctional protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. The expression of HBx in C. elegans induced apoptosis and necrosis, which mimics one of the early cellular events following liver infection by HBV. HBx-induced cell death pathways involved cell death regulators and executors and components of the necrosis pathway. The Nef protein regulates multiple functions in the host that enhance HIV-1 pathogenesis. The feeding of worms on bacteria transformed with a sequence of Nef resulted in its absorption via the gut. Nef induced several altered physiological effects in animals such as lipodystrophy, impaired locomotion, compromised reproductive performance and reduced life span. These physiological effects were comparable to with pathologic symptoms in mammals.
Feeding worms with a viral protein
[46]
[34, 48, 54]
[38]
[49]
[50]
[51]
[52]
a Abbreviations: AcMNPV, Autographa californica multiple nucleopolyhedrovirus; FHV, Flock House virus; HBV, hepatitis B virus; HDV, hepatitis D virus; HIV-1, human immunodeficiency virus 1; OrV, Orsay virus; VSV, vesicular stomatitis virus; VV, vaccinia virus
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biogenesis of miRNAs and siRNAs, respectively [28]. Unlike insects and plants, which dedicate the entire RNAi machinery to the antiviral response, worms use it for the regulation of physiological processes as well [29]. For example, mutations in ego-1 disrupt RNAi for some germline genes and lead to defects in germline development, and animals with a defective dcr-1 gene are sterile [25, 30]. In these physiological events, DCR-1 processes stem-loop structures that are characteristic of precursor microRNAs (pre-miRNAs) to generate mature microRNAs (miRNAs), apparently without a requirement for other factors [31]. DCR-1 also acts on both exogenous (dsRNA microinjection or feeding) and endogenous double-stranded RNAs (aberrant endogenous dsRNA) (Fig. 1). For exogenous dsRNA, the process requires, aside from DCR-1, the binding of the RNA-binding protein RDE-4 [32]. During viral infection, similar components are recruited, but the process is initiated by an RIG-1 homologue called DHR-1. DHR-1 recruits DCR-1 and its partner RDE-4 to the viral dsRNA replication intermediate. The processing by DCR-1 generates primary siRNAs that then bind to the Argonaute protein RDE-1. Mutation of Rde-1 leads to higher viral loads, confirming the role of the RNAi system as an antiviral mechanism [33, 34]. After binding to RDE-1, the primary siRNAs can be amplified by RNA-dependent RNA Fig. 1 Description of the main components of the C. elegans RNAi machinery and differences compared to other exogenous sources of dsRNA
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polymerases to generate antisense secondary siRNAs [12]. This process is mediated by primary siRNAs that prime the synthesis of dsRNA using the target mRNA itself as a template. Thus, secondary siRNAs are encoded by the target gene but not by the trigger RNA that originated the primary siRNAs. This amplification process, which generates a systemic effect, does not occur in mammals [12]. Secondary siRNAs target the viral genome and are thus called viRNAs [35]. This targeting of the viral RNA by viRNAs is the signal for the destruction of the molecule by specific AGO proteins (SAGO-2) [36].
Strategies to study viruses in C. elegans Infection of C. elegans cells in vitro In this approach, normal and RNAi-defective primary embryonic C. elegans cells were infected with a recombinant vesicular stomatitis virus (VSV, family Rhabdoviridae), Indiana strain, carrying a GFP-P gene fusion protein to facilitate evaluation (Table 1) [37]. The embryonic cell culture was produced from eggs purified from bleached gravid worms (bleach is used to eliminate contaminant bacteria and to synchronize worm cultures). Cells obtained from eggs after enzymatic treatment were seeded at
The nematode Caenorhabditis elegans
3 9 106 cells per cm2 in eight-well chambered coverglasses coated with peanut lectin and maintained at 27 °C in a sealed humid chamber until infection. Twenty to twenty-four hours after exposure to virus at different moi, infection was detected through the presence of green fluorescent cells; infectious virus from infected worm cells was evaluated by plaque assays on Vero cells, and the relative concentrations of VSV negative-strand RNA were measured by quantitative PCR of cDNA. To evaluate the ability of the VSV virus to spread among cells, a neutralizing antibody targeting the viral attachment protein was used to treat infected cells. Using this approach, a reduction of the number of infected cells was observed, suggesting that the virus was able to spread from cell to cell. After seven days of infection, cells with a defective RNAi apparatus (mutations in rde-1 and rde-4 genes) produced more GFP, genomic minus RNA, and infectious particles than uninfected cells. When using C. elegans strains with enhanced RNAi machinery (like, for example, cells defective in nucleases that normally degrade siRNAs), the result was the opposite. These experiments showed that resistance to VSV infection correlated with a normal RNAi machinery, demonstrating the importance of this mechanism in the antiviral response.
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In this approach, segments of Flock House virus (FHV) (family Nodaviridae), which has a bipartite positive-sense RNA genome, were introduced into C. elegans by microinjection [38] (Table 1, Fig. 2). When a DNA fragment is microinjected into worm gonads, it is maintained in extrachromosomal arrays in the generated progeny and is expressed according to the promoter that is selected.
FHV was chosen in this work because it could replicate in different types of cells (plant, yeast, insect and mammalian) [39]. Segment 1 (RNA1) encodes the viral RNAdependent RNA polymerase, and it can replicate independently of segment 2 (RNA2), which encodes the viral capsid protein precursor. At its 30 end, FHV RNA1 encodes a subgenomic RNA3 containing two overlapping open reading frames (ORFs) encoding proteins B1 and B2. B2 is an RNAi suppressor that is active across the animal and plant kingdoms [40]. Transgenic strains of C. elegans carrying these segments under the control of a heat-inducible promoter generated all of the transcripts produced during normal viral replication. The detection of self-replication of RNA1 and transreplication of RNA2 together with the production of subgenomic RNA was taken as evidence of viral replication, although no viral particles were shown to be present. In addition, it was observed that the replication of the virus was inhibited via an RNAi mechanism mediated by the B2 protein. When the B2 gene was mutated, viral replication increased [38]. In a later study, the FHV system was utilized to identify genes that are essential for the antiviral response, such as the previously mentioned RDE-1 protein, which was found to be essential for siRNAs but not for miRNAs [26]. In turn, the broad-spectrum RNAi inhibitor FHV B2 protein was found to act upstream of rde-1 by targeting the virusderived primary siRNAs and facilitating infection of C. elegans by, for example, Orsay virus [41] (see below). In spite of these findings, this approach does not allow the host response to the whole virus to be studied, since a real infection is not happening. As a consequence, some details of the replication cycle and the ability of the virus to propagate in other hosts cannot be studied.
Fig. 2 Genomic characteristics of the Flock House virus (FHV) genome. Segment 1 (RNA1) encodes the RNA-dependent RNA polymerase; segment 2 (RNA2) encodes the capsid protein. The B2
protein is produced from a subgenomic RNA3. RNA1 and RNA 2 were cloned downstream of a heat-inducible promoter and microinjected into C. elegans gonads to obtain transgenic strains
Transgenic C. elegans expressing a fragment of a viral genome
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In vivo infection of C. elegans with mammalian viruses
Natural infection of C. elegans by viruses
By utilizing polyethylenglycol (PEG) to make the cuticle permeable, C. elegans larva (L3/L4 stages) could be infected with a recombinant vaccinia virus (VV, family Poxviridae) expressing b-galactosidase/CAT (chloramphenicol acetyltransferase) and with a recombinant Sindbis virus expressing GFP (green fluorescent protein). PEG was used to facilitate viral entry, since natural infection with these viruses could not be achieved (Table 1). The CAT gene was cloned under the control of an early VV promoter, whereas the b-galactosidase gene was cloned under the control of a late one; both genes were only turned on if the recombinant virus was undergoing productive genome replication. Thus, the level of expression of X-gal and bgalactosidase was taken as a measure of the level of viral replication. This was also analyzed by real-time PCR and western blotting. Parameters such as viral titers, PEG size and concentration, incubation time and temperature, and the developmental stage of the worms were tested. It was found that 5 % of early adults could be infected with 1 9 107 PFU /ml in the presence of 2 % PEG 1000 for 5 minutes at room temperature. The percentage could be increased to 18 % by moving the worms to a buffer (M9) with the same viral concentration for 6 hours at room temperature. Viral particles were observed by electron microscopy, and viable virus could be recovered from several worm tissues. Blue X-Gal staining was observed in many tissues and organs, including pharynx, intestine, muscle, hypodermis, and occasionally germline. Parameters of infection could only be detected for 3 days postinfection (p.i.), suggesting that some restriction to VV replication existed in the wt worm. By day 4, 80 % of the infected worms had died, whereas there were no dead animals when infection was performed with UV-treated virus. Next, the authors investigated the role of programmed cell death in the observed restriction of viral infection. In C. elegans, four genes control programmed cell death: ced3, ced-4, ced-9 and egl-1. The first three encode proteins similar to human caspases, Apaf-1 and Bcl-2, respectively [42–44], whereas EGL-1 is a small BH3-containing protein [45]. Mutations in all of these genes block most of the programmed cell death, and fewer apoptotic bodies can then be seen during the development of the worm. This is quite evident in the pharynx, where more than 80 % of the cells that normally die survive. By means of different C. elegans strains mutated in this apoptotic pathway, the authors found a role for caspases inhibiting infection [46]. All of the findings described for VV infection were also observed with the recombinant Sindbis virus expressing GFP.
Very recently, two new RNA viruses with similarities to members of the family Nodaviridae were described in natural populations of Caernorhabditis worms. One of them (Orsay virus) infected C. elegans naturally, whereas Santeuil virus could do the same in C. briggsae, another worm species from the same genus as C. elegans (Table 1) [34]. Both of them were identified by a pyrosequencing approach from animals with abnormal morphologies in intestinal cells (storage granules disappeared and cytoplasm became fluid) in which small virus-like particles of approximately 20 nm diameter were visible by electron microscopy. As described previously, viruses in this family have a bipartite genome, with segment one (RNA1) encoding the RNA-dependent RNA polymerase and segment two (RNA2) encoding the capsid protein. Some members of this family, as shown for FHV, encode proteins B1 and B2 from a subgenomic RNA produced from RNA1. The putative RNA-dependent RNA polymerase and capsid proteins of Orsay virus shared around 30 % amino acid sequence identity with similar genes of other nodaviruses, whereas the identity between the Orsay and Santuiel virus RNA-dependent RNA polymerases was only of 44 %. Interestingly, both viruses were predicted to encode a second ORF (ORF d) in the 30 half of the RNA2 segment in addition to the capsid protein at the 50 end of the same fragment. Also, neither virus appeared to encode a B1 or a B2 protein. These features suggested that the genomic organization of these viruses was distant from that of other members of the same family. Furthermore, phylogenetic analysis of the predicted RNA polymerase and capsid proteins of both viruses showed that they were highly divergent from those of other nodaviruses, suggesting that these viruses might represent a novel genus. In vivo studies with these viruses showed that they were not integrated into the nematode genome and that they could not be transmitted vertically in spite of being present in worm cultures for longer than 50 generations (6 months). These results suggested that horizontal transmission of the virus occurs, and that these nematodes are a suitable model for the study of a virus in its natural host. In addition, C. elegans mutants deficient in the RNAi machinery (red-1, rde-4) could be infected more easily than the wild type (N2 Bristol strain), suggesting again an antiviral role of the RNAi response. These mutants showed higher levels of virus replication and more symptoms [34]. Furthermore, it was observed that natural isolates of C. elegans showed distinctive susceptibility to infection with Orsay virus, a sign of natural variation in host antiviral defenses.
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Recently, by high-throughput sequencing of wild Caenorhabditis strains, a new virus was identified in a C. briggsae isolate. This virus, called Le Blanc, was found to be distantly related to Orsay virus and Santeuil virus but still showed an overall similarity to nodaviruses [47]. More recently, studying the whole transcriptome in response to infection with Orsay virus, a significant number of virus-specific immune response genes were identified in N2 and rde-1 worms [48]. In particular, the RNAi response to the viral infection was found to be mediated by a redirection of the Argonaute protein RDE-1, leading to loss of repression of its targets. Thus, the authors suggested that the antiviral RNAi response was regulated by a competition between exogenous and endogenous RNAi pathways. Suppressors of the C. elegans RNAi response have recently been identified using the Orsay virus infection approach. In this way, the role of the FHV B2 protein as a broad-spectrum RNAi inhibitor was confirmed. Furthermore, it was found that the B2 protein acted upstream of rde-1 by targeting the virus-derived primary siRNAs and thus facilitating infection of C. elegans by Orsay virus [41]. C. elegans for the study of viral genes One of the first and few published works using C. elegans to evaluate viral genes involved the study of a baculovirus antiapoptotic gene (p35) (Table 1). When worms expressing this gene under a heat shock protein promoter were exposed to heat, there was a reduction in programmed cell death during development. This effect was observed in the embryo as a decreased number of apoptotic bodies and in the adult, as described before, in the form of extra cells in the pharynx (Table 1) [49]. In a more recent work, C. elegans was used to express a multifunctional protein from hepatitis delta virus (HDAg) [50]. HDAg is involved in the pathogenesis of this viral infection, contributing by still unknown mechanisms, to the carcinogenic process developed in hepatocytes. Typical C. elegans characteristics such as sterility, growth, and brood size were evaluated in transgenic strains expressing HDAg under the control of two different promoters, a ubiquitous promoter (fib-1) and a muscle-specific promoter (myo-2) that is only active in the pharynx. Ubiquitous expression of HDAg resulted in 20 to 70 % sterility, whereas transgenic worms expressing the protein in pharynx tissue were arrested at larval stage 2 or 3, with a reduction in brood size. Growth retardation was suggested to be explainable by possible changes in two pathways, TGF-beta and insulin-like. Transgenic C. elegans strains expressing the HBx protein of hepatitis B virus also showed a reduction in apoptotic as well as necrotic processes, similar to what is
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observed in the early stages of liver infection by this virus (Table 1) [51]. In an interesting approach, C. elegans was fed with bacteria expressing the human immunodeficiency virus Nef protein, which is known to be involved in pathogenesis by enhancing viral replication (Table 1). A phenotype with features similar to those of the in vivo infection was obtained. For example, marked lipodystrophy, neuromuscular dysfunction, impaired fertility, and reduced longevity were observed in these worms [52].
Concluding remarks This review attempts to summarize the first experiences in the use of C. elegans as a model to study viral pathogenesis. Firstly, C. elegans is a cheap model that requires minimal equipment and reagents and is only fed with bacteria. Phenotypes can be evaluated very quickly, since the worm becomes adult in 3-4 days. The similarities between C. elegans and higher eukaryotic pathways together with the availability of many mutants in those pathways represent one of its major benefits. In fact, infection by different kinds of bacterial pathogens has been analyzed in this way, allowing the identification of several genes involved in virulence and host defense. On the other hand, unlike bacteria and fungi, the worm cannot be infected very easily with foreign viruses. The diversity of the above-mentioned strategies reflects efforts to establish C. elegans as a model for infection. An artificial infection method has been based on introducing a partial virus genome of Flock House virus into animals [38, 53]. This experimental system can only examine replication of the viral RNA and is fundamentally unable to address the host response to other critical aspects of the virus life cycle such as a virus entry, virion assembly or egress [53]. As mentioned in this review, the only successful artificial infection utilized a chemical treatment (PEG) to infect C.elegans with vaccinia virus [46]. However, no other papers have been published using this approach, or other new ones, suggesting that the worm cuticula may represent a major barrier to these studies. Certainly, the PEG approach makes the study of particular aspects of the viral replicative cycle such as receptor recognition and entry very difficult. Another level of difficulty is added if, for example, transgenic strains have to be produced for expression of particular viral genes. This approach requires expertise in microinjection and a good knowledge of worm genetics. If it can be achieved, however, the effect of many viral genes can be analyzed in the background of different mutations involving highly conserved pathways. Furthermore, because C. elegans is transparent, the progression of
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infection with a marker virus or the expression of a particular viral gene in a specific tissue can be easily monitored. The discovery of nodaviruses that naturally infect C. elegans and its close relative C. briggsae, has generated new expectations. This virus has shown the ability to be transmitted horizontally, causing major damage in intestinal cells [46, 53]. Nodavirus infections are therefore becoming a feasible system for studies on viral-host interaction, specificity, immune response, pathogenesis and evolution. The finding of natural infections in C. elegans will certainly provide a deeper comprehension of the viral life cycle.
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