Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus

Abstract

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

FIG. 1

Clinical course of rabbits at 10 days postinoculation. Rabbits infected with the wild-type MV (A and B) show secondary skin lesions turning necrotic on the head, the back, and the legs and edema of the testicles (A) and show primary and secondary myxomas on the ears, leading to abnormal posture, and on the nose and around the eyes, and blepharoconjunctivitis (B). Rabbits infected with the MV-Serp2⁻ mutant (C and D) show no secondary skin lesions and little edema of the genital region (C); they also show marked diffuse inflammation of the ears and blepharoconjunctivitis (D).

FIG. 2

Hematoxylin and eosin-stained skin samples from rabbits at 11 days postinoculation with wild-type MV (A and C) or with MV-Serp2⁻ mutant (B and D). Magnification: ×40 (A and B); ×200 (C and D). The panels show perivascular dermatitis with diffuse edema, thrombosis and perivascular accumulation of heterophils (A); perivascular dermatitis with a less-severe edema and interstitial infiltrates of mononuclear cells (B); severe perivascular and interstitial accumulation of heterophils (C); and interstitial infiltrates of mononuclear cells (C).

FIG. 3

Parotid lymph node (TUNEL method). The panels show samples at 8 days postinoculation with the wild-type MV, with minimal lymphocytic apoptosis (A), and infection with the MV-Serp2⁻ mutant at 4 days postinoculation, with focal extensive lymphocytic apoptosis in cortical areas (arrows) (B); at 4 days postinoculation, with focal extensive lymphocytic apoptosis at a higher magnification (C); and at 11 days postinoculation, with remnants of apoptotic foci in a lymphodepleted parenchyma (arrow) (D). Magnification: ×100 (A); ×40 (B); ×100 (C); ×40 (D).

FIG. 4

Parotid lymph node (immunohistochemistry) from rabbits at 8 days postinoculation (×400). Infected cells are visualized with DAB (arrow). (A) Infection with the wild-type MV. The cells present in the lymph node are mostly histiocytes and activated fibroblasts (large nucleus, double arrows). These and other cells are infected. (B) Infection with the MV-Serp2⁻ mutant. Most cells are infected, and mononuclear cells are predominant.