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

Abstract

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.

Keywords: Caenorhabditis elegans; Endoplasmic reticulum stress; Gonad retardation; Green fluorescence proteins; Hepatitis B virus; Surface antigens.

Figure 1

Expression of various lengths of hepatitis B virus antigens in whole worms induced defects in the rate of egg-laying. A-F: Micrographs of transgenic worms expressing LHBsAg (A and B), MHBsAg (C and D), and SHBsAg (E and F) were captured under a bright-field microscope (A, C, and E) and a fluorescence microscope (B, D, and F). The heads of the worms are shown toward the left. The scale bar indicates 200 μm. G: Egg-laying capability of three lines of transgenic worms and wild-type worms (N2) shown using various color bars. The rate of egg-laying in 3 to 7 d post-hatching is shown above the bar. HBsAgs: Hepatitis B virus antigens.

Figure 2

Differential interference contrast micrographs of gonad development in transgenic worms at various post-hatching times. The upper row shows a wild-type worm (N2); the second row shows a transgenic worm expressing SHBsAg; the third row shows a transgenic worm expressing MHBsAg; and the bottom row shows a transgenic worm expressing LHBsAg. The right column shows the gonads at 52 h post-hatching and the left column at shows gonads at 68 h post-hatching expect the worm expressing SHBsAg is at 72 h post-hatching. The gonad contour is indicated with dotted lines and the tip (distal end) is marked by asterisk.

Figure 3

Reverse transcription polymerase reaction analyses of mRNA levels in various transgenic worms. The four transcripts (enpl-1, hsp-4, pdi-3, and xbp-1), ER-stress markers, from wild-type worms (N2) with or without DTT pretreatment, an ER stress inducer, and three lines of transgenic worms were analyzed using RT-PCR and gel-electrophoresis. The transcript of the translation factor (eft-2) served as a loading control. N2 worms treated with and without DTT and transgenic worms are indicated above the gel. DTT-treated worms severed as positive controls of ER-stress responses. DTT: Dithiothreitol; RT-PCR: Reverse transcription polymerase reaction.

Figure 4

Features of transgenic worms expressing SHBsAgs in different tissues. A-F: Micrographs of transgenic worms expressing SHBsAgs in the pharynx (A, and B), intestinal cells (C and D), and neurons (E and F) were captured under a bright-field microscope (A, C, and E) and a fluorescence microscope (B, D, and F). The heads of the worms are shown toward the left. The scale bar indicates 200 μm; G: Gonad development in transgenic worms: The upper row is a transgenic worm expressing SHBsAg in the pharynx; the middle row is a transgenic worm expressing SHBsAg in intestinal cells; and the lower row is a transgenic worm expressing SHBsAg in neurons. Images were captured under a DIC microscope. The right column shows the gonads at 52 h post-hatching and the left column at 68 h post-hatching. The gonad contour is outlined by dotted lines and the tip (distal end) is marked by asterisk. HBsAgs: Hepatitis B virus antigens.