Restriction of vaccinia virus replication by a ced-3 and ced-4-dependent pathway in Caenorhabditis elegans

Abstract

Genetic tractability and easy manipulation make Caenorhabditis elegans a good model to study host-pathogen interactions. Dozens of different bacterial species can pathogenically infect C. elegans under laboratory conditions, and all of these microbes are extracellular pathogens to nematodes. Viruses, on the other hand, are obligate intracellular parasites, and yet no viral infections have been reported for C. elegans. We established a procedure allowing vaccinia virus to enter and subsequently replicate in C. elegans. 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 (PCD) genes ced-3, ced-4, ced-9, and egl-1 control vaccinia virus replication in C. elegans. The ability of ced-3 and ced-4 alleles to restrict virus replication is correlated with their cell-killing activities. Moreover, the increase in vaccinia virus replication levels in the PCD-defective mutants was not likely to be caused by the extra live cells, as neither the inhibition of PCD by icd-1 overexpression nor the presence of extra cells after extra cell divisions in cul-1 or lin-23 mutants had any significant effect on vaccinia virus replication. Therefore, the core PCD genes possess a unique function in controlling vaccinia virus replication in C. elegans.

Fig. 1.

Replication of VV in PEG-treated C. elegans. Groups of about 1000 worms at L3-L4 stage were subjected to PEG-mediated infection by 1 × 10⁷ pfu/ml of vaccinia virus (VV). (A) Wild-type worms exposed to 2% PEG alone (Upper) or to 2% PEG plus 1 × 10⁷ pfu/ml VV (Lower) on the second day postinfection (p.i.) were fixed and stained with X-Gal. (B) PCR analysis. Accumulation of VV genomic DNA was analyzed by PCR in wild-type N2 and ced-3(n717) mutant worms at day 0 or day 3 p.i. (C) Real-time PCR analysis. The relative replication level of VV genomic DNA in wild-type N2 or ced-3(n717) mutant worms was determined by real-time PCR using primers specific for VV B5R gene. The result represents the abundance of replicative DNA in the sample collected at day 3 relative to that at day 0 p.i. The Ct values are shown separately in Table 2, which is published as supporting information on the PNAS web site. All experiments were repeated three times independently and data are means ± SD. (D) Immunoblot. The amounts of VV proteins expressed in N2 and ced-3(n717) mutant at the indicated time points were analyzed by immunoblotting using antibody specific for VV structural D8L protein. For simplicity, we defined day 0 as the time when the 5-min virus exposure with PEG followed by the 6-h virus treatment without PEG had been completed.

Fig. 2.

The PCD mutations enhance VV replication in PEG-treated C. elegans. About 1,000 worms (stages L3-L4) of each strain were infected with vaccinia virus in the presence of PEG. (A) VV titers in lysates of ced-3(n717) and ced-4(n1162) mutant strains were measured for β-galactosidase activity at OD₆₆₀ at different days p.i. (B) After infection, the titers of VV from each C. elegans strain were determined at the indicated days by the standard plaque-forming assay. (C) Virus replication levels in the ced-9(n1950) and egl-1(n1084n3082) mutant strains were determined from the spectrophotometric β-galactosidase activity at OD₆₆₀ at the indicated times. All experiments were repeated three times independently (∗, P < 0.05; ∗∗, P < 0.01 by Student’s t test). Data are means ± SD.

Fig. 3.

The effects of different ced-3 and ced-4 alleles on vaccinia virus replication in C. elegans. About 1,000 worms (stages L3–L4) of the indicated strains were infected with VV in the presence of PEG. The level of virus replication in each strain was determined from the β-galactosidase activity at OD₆₆₀. (A) Viral replication levels in most of the ced-3 mutants tested were higher than those in wild type. (B) VV replication levels in ced-4 mutants were significantly higher than those in wild-type worms at the second day p.i. (C) Overexpression of icd-1 or mutations in ces-1, ces-2, cul-1, and lin-23 did not affect VV titers. All experiments were repeated three times. (∗, P < 0.05; ∗∗, P < 0.01 by Student’s t test). Data are means ± SD.

Fig. 4.

Vaccinia-mediated killing of C. elegans is independent of ced-3 and ced-4. About 100 worms (stages L3–L4) of each C. elegans strain were treated under the indicated conditions. Dead worms were picked on indicated days and tested by using X-Gal staining. The percentage of the dead infected worms of each strain was calculated by the following formula: (the number of total X-Gal-positive worms that died by the indicated days/the number of total infected worms) × 100%. The number of total infected worms was calculated by the number of total animals times the infection rate. (A) No PEG or virus treatment. (B) PEG treatment without virus. (C) PEG plus virus treatment. All data (means ± SD) were obtained from four independent experiments.