Granzymes and caspase 3 play important roles in control of gammaherpesvirus latency

Abstract

Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (gammaHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined gammaHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating gammaHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.

FIG. 1.

Granzymes A and B have functionally redundant roles in regulating γHV68 early latent infection. Peritoneal cells from infected GzmA^−/−, GzmB^−/−, GzmAXB^−/−, and 129/SvJ mice were analyzed for the frequency of cells which reactivated virus ex vivo and which harbored viral genome at 16 (A) or 42 (B) dpi. No preformed infectious virus was detected in mechanically disrupted samples (data not shown). Number of independent experiments: GzmA^−/−, three; GzmB^−/−, four; GzmAXB^−/−, four; 129/SvJ, seven.

FIG. 2.

Orphan granzymes in the GzmB locus regulated γHV68 latent infection at the early time point. Peritoneal cells from infected mice were analyzed for the frequency of cells which reactivated virus ex vivo and which harbored viral genome at 16 dpi. (A) Comparison of GzmB-cluster^−/−, GzmB^−/−, and 129/SvJ mice. (B) Comparison of GzmAXB-cluster^−/−, GzmAXB^−/−, and 129/SvJ mice. No preformed infectious virus was detected in mechanically disrupted samples (data not shown). Number of independent experiments: GzmB-cluster^−/−, five; GzmB^−/−, four; GzmAXB-cluster^−/−, five; GzmAXB^−/−, four; 129/SvJ, seven.

FIG. 3.

Orphan granzymes, but not granzymes A and B, regulated γHV68 latent infection at the late time point. Peritoneal cells from infected GzmAXB^−/−, GzmB-cluster^−/−, GzmAXB-cluster^−/−, and 129/SvJ mice were analyzed at 42 dpi for the frequency of cells which reactivated virus ex vivo (A) or cells which harbored viral genome (B). No preformed infectious virus was detected in mechanically disrupted samples (data not shown). Number of independent experiments: GzmB-cluster^−/−, five; GzmAXB-cluster^−/−, four; GzmAXB^−/−, four; 129/SvJ, eight.

FIG. 4.

Impaired regulation of latent infection in granzyme-deficient mice is not due to uncontrolled lytic replication during acute infection. Titers in spleens from infected GzmAXB^−/−, GzmB-cluster^−/−, GzmAXB-cluster^−/−, and 129/SvJ mice were determined by plaque assay at 4 (A) or 9 (B) dpi. The limit of detection is indicated by the horizontal line. The number of mice analyzed is given above each bar.

FIG. 5.

Caspase 3-dependent processes regulated γHV68 chronic infection at the early time point. Peritoneal cells from infected caspase 3^−/− and B6 mice were analyzed for the frequency of cells which reactivated virus ex vivo and which harbored viral genome at 16 (A) or 42 (B) dpi. No preformed infectious virus was detected in mechanically disrupted samples (data not shown). Number of independent experiments: caspase 3^−/−, four; B6, four.