Defects in CISD-1, a mitochondrial iron-sulfur protein, lower glucose level and ATP production in Caenorhabditis elegans.

BACKGROUND: CDGSH iron sulfur domain-containing protein 1 (CISD-1) belongs to the CISD protein family that is evolutionary conserved across different species. In mammals, CISD-1 protein has been implicated in diseases such as cancers and diabetes. As a tractable model organism to study disease-associated proteins, we employed Caenorhabditis elegans in this study with an aim to establish a model for interrogating the functional relevance of CISD-1 in human metabolic conditions. METHODS: We first bioinformatically identified the human Cisd-1 homologue in worms. We then employed N2 wild-type and cisd-1(tm4993) mutant to investigate the consequences of CISD-1 loss-of-function on: 1) the expression pattern of CISD-1, 2) mitochondrial morphology pattern, 3) mitochondrial function and bioenergetics, and 4) the effects of anti-diabetes drugs. RESULTS: We first identified C. elegans W02B12.15 gene as the human Cisd-1 homologous gene, and pinpointed the localization of CISD-1 to the outer membrane of mitochondria. As compared with the N2 wild-type worm, cisd-1(tm4993) mutant exhibited a higher proportion of hyperfused form of mitochondria. This structural abnormality was associated with the generation of higher levels of ROS and mitochondrial superoxide but lower ATP. These physiological changes in mutants did not result in discernable effects on animal motility and lifespan. Moreover, the amount of glucose in N2 wild-type worms treated with troglitazone and pioglitazone, derivatives of TZD, was reduced to a comparable level as in the mutant animals. CONCLUSIONS: By focusing on the Cisd-1 gene, our study established a C. elegans genetic system suitable for modeling human diabetes-related diseases.

Nucleolar control by a non-apoptotic p53-caspases-deubiquitinylase axis promotes resistance to bacterial infection.

The nucleolus is best known for its cellular role in regulating ribosome production and growth. More recently, an unanticipated role for the nucleolus in innate immunity has recently emerged whereby downregulation of fibrillarin and nucleolar contraction confers pathogen resistance across taxa. The mechanism of this downregulation, however, remains obscure. Here we report that rather than fibrillarin itself being the proximal factor in this pathway, the key player is a fibrillarin-stabilizing deubiquitinylase USP-33. This was discovered by a candidate-gene search of Caenorhabditis elegans in which CED-3 caspase was revealed to execute targeted cleavage of USP-33, thus destabilizing fibrillarin. We also showed that cep-1 and ced-3 mutant worms altered nucleolar size and decreased antimicrobial peptide gene, spp-1, expression rendering susceptibility to bacterial infection. These phenotypes were reversed by usp-33 knockdown, thus linking the CEP-1-CED-3-USP-33 pathway with nucleolar control and resistance to bacterial infection in worms. Parallel experiments with the human analogs of caspases and USP36 revealed similar roles in coordinating these two processes. In summary, our work outlined a conserved cascade that connects cell death signaling to nucleolar control and innate immune response.

Reflex and habituation behavior of Caenorhabditis elegans assessed by a mechanical vibration system and image analysis.

BACKGROUND: The nematode Caenorhabditis elegans is an emerging invertebrate animal model for investigating neuronal functions in behavioral assays. C. elegans mechanosensation was characterized by the use of a constant mechanical stimulation transmitter followed by quantitative imaging. NEW METHOD: C. elegans reflex and habituation behaviors were characterized by mechanical vibration followed by image analysis. A custom-designed system consists of an aluminum alloy Petri dish holder frame coupled with a mechanical vibration buzzer delivering adjustable pulsed vibration to an agar plate. The basal and evoked movements of C. elegans were recorded by a microscopic digital camera followed by quantitative analysis using microscopic imaging software. RESULTS: Application of the platform in C. elegans was demonstrated with three proof-of-concept experiments: (1) Evaluation of the reflex response stimulated by tapping and mechanical vibration with a mechano-sensation defective mutant. (2) Comparison of the reflex response stimulated by mechanical vibration between wild type and aging mutants. (3) Assessment of the efficacy of the mechanical vibration system on long-term memory for habituation. COMPARISON WITH EXISTING METHODS: Conventional C. elegans mechanosensation techniques depend on stimulation either by manually touching a single animal or tapping the Petri dish followed by scoring via visual observation from the examiner. The mechanical vibration method has greater capacity compared to conventional methods which are labor-intensive, have low throughput and lack quantifiable parameters. CONCLUSIONS: The mechanical vibration system followed by image analysis is a convenient and integrated platform for investigatingC. elegans reflex and habituation in aging and neural behavioral assays.

IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans.

NADPH is a reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH-producing enzymes predisposes cells to growth retardation and demise. It was hypothesized that double deficiency of the NADPH-generating enzymes, GSPD-1 (Glucose-6-phosphate 1-dehydrogenase), a functional homolog of human glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and IDH-1 (isocitrate dehydrogenase-1) affect growth and development in the nematode, Caenorhabditis elegans (C. elegans). The idh-1;gspd-1(RNAi) double-deficient C. elegans model displayed shrinkage of body size, growth retardation, slowed locomotion, and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by severe NADPH insufficiency by the idh-1;gspd-1(RNAi) double-deficiency. The principal component analysis (PCA) points to a distinct metabolomic profile of idh-1;gspd-1(RNAi) double-deficiency. Further metabolomic analysis revealed that NADPH-dependent and glutamate-dependent amino acid biosynthesis were significantly affected. The reduced pool of amino acids may affect protein synthesis, as indicated by the absence of NAS-37 expression during the molting process. In short, double deficiency of GSPD-1 and IDH-1 causes growth retardation and molting defects, which are, in part, attributed to defective protein synthesis, possibly mediated by altered amino acid biosynthesis and metabolism in C. elegans.

Size scaling of nucleolus in Caenorhabditis elegans embryos.

Nucleolus is viewed as a plurifunctional center in the cell, tightly linked to ribosome biosynthesis. As a non-membranous structure, how the size of nucleolus is determined is a long outstanding question, and the possibility of "direct size scaling to the nucleus" was raised by genetic studies in fission yeast. Here, we used the model organism Caenorhabditis elegans to test this hypothesis in multi-cellular organisms. We depleted ani-2, ima-3, or C27D9.1 by RNAi feeding, which altered embryo sizes to different extents in ncl-1 mutant worms. DIC imaging provided evidence that in size-altering embryo nucleolar size decreases in small cells and increases in large cells. Furthermore, analyses of nucleolar size in four blastomeres (ABa, ABp, EMS, and P2) within the same embryo of ncl-1 mutants consistently demonstrated the correspondence between cell and nucleolar sizes - the small cells (EMS and P2) have smaller nucleoli in comparison to the large cells (ABa).

Expression of hepatitis B virus surface antigens induces defective gonad phenotypes in Caenorhabditis elegans.

AIM: To test whether a simple animal, Caenorhabditis elegans (C. elegans), can be used as an alternative model to study the interaction between hepatitis B virus antigens (HBsAg) and host factors. METHODS: Three plasmids that were able to express the large, middle and small forms of HBsAgs (LHBsAg, MHBsAg, and SHBsAg, respectively) driven by a ubiquitous promoter (fib-1) and three that were able to express SHBsAg driven by different tissue-specific promoters were constructed and microinjected into worms. The brood size, egg-laying rate, and gonad development of transgenic worms were analyzed using microscopy. Levels of mRNA related to endoplasmic reticulum stress, enpl-1, hsp-4, pdi-3 and xbp-1, were determined using reverse transcription polymerase reaction (RT-PCRs) in three lines of transgenic worms and dithiothreitol (DTT)-treated wild-type worms. RESULTS: Severe defects in egg-laying, decreases in brood size, and gonad retardation were observed in transgenic worms expressing SHBsAg whereas moderate defects were observed in transgenic worms expressing LHBsAg and MHBsAg. RT-PCR analysis revealed that enpl-1, hsp-4 and pdi-3 transcripts were significantly elevated in worms expressing LHBsAg and MHBsAg and in wild-type worms pretreated with DTT. By contrast, only pdi-3 was increased in worms expressing SHBsAg. To further determine which tissue expressing SHBsAg could induce gonad retardation, we substituted the fib-1 promoter with three tissue-specific promoters (myo-2 for the pharynx, est-1 for the intestines and mec-7 for the neurons) and generated corresponding transgenic animals. Moderate defective phenotypes were observed in worms expressing SHBsAg in the pharynx and intestines but not in worms expressing SHBsAg in the neurons, suggesting that the secreted SHBsAg may trigger a cross-talk signal between the digestive track and the gonad resulting in defective phenotypes. CONCLUSION: Ectopic expression of three forms of HBsAg that causes recognizable phenotypes in transgenic worms suggests that C. elegans can be used as an alternative model for studying virus-host interactions because the resulting phenotype is easily detected through microscopy.

Impaired embryonic development in glucose-6-phosphate dehydrogenase-deficient Caenorhabditis elegans due to abnormal redox homeostasis induced activation of calcium-independent phospholipase and alteration of glycerophospholipid metabolism.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A2 (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

Lipidomic Analysis of Caenorhabditis elegans Embryos.

Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is simple and homogeneous. Here, we demonstrate detailed procedures of C. elegans culture, embryo isolation, lipid extraction and metabolomic data analysis.

Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging.

Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

Genetic control of nucleolar size: An evolutionary perspective.

Exploiting a C. elegans mutant (ncl-1) exhibiting nucleolar abnormalities, we recently identified the let-7/ncl-1/fib-1 genetic cascade underlying proper rRNA abundance and nucleolar size. These 3 factors, let-7 (a miRNA), NCL-1 (a member of the TRIM-NHL family), and fibrillarin (a nucleolar methyltransferase), are evolutionarily conserved across metazoans. In this article, we provide several lines of bioinformatic evidence showing that human and Drosophila homologues of C. elegans NCL-1, TRIM-71 and Brat, respectively, likely act as translational suppressors of fibrillarin. Moreover, since their 3'-UTRs contain putative target sites, they may also be under the control of the let-7 miRNA. We hypothesize that let-7, TRIM and fibrillarin contribute activities in concert, and constitute a conserved network controlling nucleolar size in eukaryotes. We provide an in-depth literature review of various molecular pathways, including the let-7/ncl-1/fib-1 genetic cascade, implicated in the regulation of nucleolar size.

A Genetic Cascade of let-7-ncl-1-fib-1 Modulates Nucleolar Size and rRNA Pool in Caenorhabditis elegans.

Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3'UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.

Loss of DNase II function in the gonad is associated with a higher expression of antimicrobial genes in Caenorhabditis elegans.

Three waves of apoptosis shape the development of Caenorhabditis elegans. Although the exact roles of the three DNase II genes (nuc-1, crn-6 and crn-7), which are known to mediate degradation of apoptotic DNA, in the embryonic and larval phases of apoptosis have been characterized, the DNase II acting in the third wave of germ cell apoptosis remains undetermined. In the present study, we performed in vitro and in vivo assays on various mutant nematodes to demonstrate that NUC-1 and CRN-7, but not CRN-6, function in germ cell apoptosis. In addition, in situ DNA-break detection and anti-phosphorylated ERK (extracellular-signal-regulated kinase) staining illustrated the sequential and spatially regulated actions of NUC-1 and CRN-7, at the pachytene zone of the gonad and at the loop respectively. In line with the notion that UV-induced DNA fragment accumulation in the gonad activates innate immunity responses, we also found that loss of NUC-1 and CRN-7 lead to up-regulation of antimicrobial genes (abf-2, spp-1, nlp-29, cnc-2, and lys-7). Our observations suggest that an incomplete digestion of DNA fragments resulting from the absence of NUC-1 or CRN-7 in the gonad could induce the ERK signalling, consequently activating antimicrobial gene expression. Taken together, the results of the present study demonstrate for the first time that nuc-1 and crn-7 play a role in degrading apoptotic DNA in distinct sites of the gonad, and act as negative regulators of innate immunity in C. elegans.

Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos.

Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulfing cells (non-autonomous). The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method is used as an in situ assay of apoptosis by labelling DNA fragments generated by caspase-associated DNase (CAD), but not those by the downstream DNase II. In the present study, we report a method of ToLFP (topoisomerase ligation fluorescence probes) for directly visualizing DNA fragments generated by DNase II in Caenorhabditis elegans embryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells in ced-1 embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals between ced-1 and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was ~3-7. Among three DNase II mutant embryos (nuc-1, crn-6 and crn-7), nuc-1 embryos exhibited the least number of ToLFP. The ToLFP results confirmed the previous findings that NUC-1 is the major DNase II for degrading apoptotic DNA. To further elucidate NUC-1's mode of action, nuc-1-rescuing transgenic worms that ectopically express free or membrane-bound forms of NUC-1 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed NUC-1 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA.

Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy.

Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

Lysine-71 in the large delta antigen of hepatitis delta virus clade 3 modulates its localization and secretion.

Hepatitis delta virus (HDV) is an RNA virus and eight clades of HDV have been identified. HDV clade 3 (HDV-3) is isolated only in the northern area of South America. The outcome of HDV-3 infection is associated with severe fulminant hepatitis. Variations in the large delta antigen (LDAg) between HDV clade 1 (HDV-1) and HDV-3 have been proposed to contribute to differences in viral secretion efficiency, but which changes might be relevant remains unclear. The control of subcellular localization of LDAg has been reported to be associated with post-translational modifications, such as phosphorylation and isoprenylation. We have observed evidence for acetylation on the LDAg of HDV-3 (LDAg-3) and LDAg of HDV-1 (LDAg-1). Green fluorescent protein-fused LDAg-3 (GFP-LD3) was used to investigate the cellular distribution and secretion of the protein. Sequence alignment of LDAg amino acids suggested that lysine-71 of LDAg-3 could be an acetylation site. Expression of a mutant form of LDAg-3 with an arginine-substitution at lysine-71 (GFP-LD3K71R) showed a distribution of the protein predominantly in the cytoplasm instead of the nucleus. Western blot analyses of secreted empty viral particles (EVPs) revealed a higher amount of secreted GFP-LD3K71R compared to GFP-LD3. Furthermore, the ectopic expression of p300, a histone acetyltransferase, led to a reduction of GFP-LD3 in EVPs. By contrast, expression of three histone deacetylases (HDAC-4, -5, and -6) facilitated the secretion of GFP-LD3. Combined, our observations support the hypothesis that the acetylation status of LDAg-3 plays a role in regulating LDAg-3's localization inside the nucleus or cytoplasm, and its secretion.

The nucleolus of Caenorhabditis elegans.

Nucleolar size and appearance correlate with ribosome biogenesis and cellular activity. The mechanisms underlying changes in nucleolar appearance and regulation of nucleolar size that occur during differentiation and cell cycle progression are not well understood. Caenorhabditis elegans provides a good model for studying these processes because of its small size and transparent body, well-characterized cell types and lineages, and because its cells display various sizes of nucleoli. This paper details the advantages of using C. elegans to investigate features of the nucleolus during the organism's development by following dynamic changes in fibrillarin (FIB-1) in the cells of early embryos and aged worms. This paper also illustrates the involvement of the ncl-1 gene and other possible candidate genes in nucleolar-size control. Lastly, we summarize the ribosomal proteins involved in life span and innate immunity, and those homologous genes that correspond to human disorders of ribosomopathy.

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion.

BACKGROUND: Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. METHODS: A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. RESULTS: Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion. CONCLUSIONS: Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity.

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors.

Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion.

Hepatitis D antigens cause growth retardation and brood-size reduction in C. elegans.

Caenorhabditis elegans is a model organism that has been used to study human bacterial and viral pathogenesis. We report here the expression of human hepatitis delta viral antigens (HDAg) in C. elegans and measure the effect on the sterility, growth, and brood size in transgenic worms. Expression of HDAg under two different promoters, fib-1 (a ubiquitous promoter) and myo-2 (a pharynx-specific promoter), was achieved in C. elegans using dicistronic or tricistronic vectors derived from the operon CEOP5428. Transgenic worms expressing HDAg ubiquitously resulted in 20% to 70% sterility while those expressing HDAg in the pharynx displayed 70% sterility. Most of worms expressing HDAg in pharynx were arrested at larvae stage 2 or 3 and displayed a 70% reduction in brood size. Domain mapping experiments suggested that the nuclear localization signal of HDAg is required for the observed effect. Heat-shock induction of HDAg expression revealed that L4 larvae were the most sensitive to brood size reduction. These studies demonstrate that C. elegans can provide an additional model for studying HDAg interactions with host targets.