Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus

https://doi.org/10.1016/j.bbapap.2020.140457Get rights and content

Highlights

  • Mice inheriting the W or P allele of the serine protease granzyme B have starkly different survival outcomes from MCMV infection

  • Differences in protease substrate preference, result in markedly different processing of host and viral proteins in infected cells

  • We pin-pointed differences in apoptotic signalling, but these did not account for the disparate outcomes of infection

  • Differences in the processing of viral proteins is likely to account for the excessive mortality of MCMV-infected GzmBW/W mice

Abstract

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

Section snippets

List of non-standard abbreviations

MCMVmouse cytomegalovirus
GzmBgranzyme B
B6inbred mouse strain C57BL/6
AADbenzyloxycarbonyl-Ala-Ala-Asp-S Benzyl
IEPDAcetyl-Ile- Glu-Pro-Asp-paranitroanilide
MOMPmitochondrial outer membrane permeabilization
tBidtruncated Bid
MHCmajor histocompatibility complex
AVannexin V
PIpropidium iodide
TAILSterminal amine isotopic labelling of substrate
MEFmouse embryonic fibroblast
CTLcytotoxic T lymphocyte
NKnatural killer
Asp-asecleavage after a P1 aspartate residue.

Mice

Typical inbred B6 mice (which have the P allele, GzmBP/P) [15] were purchased from the Walter and Eliza Hall Institute (WEHI), Melbourne, Australia. B6.GzmAB−/− and B6.GzmBW/W mice were bred and maintained at the Peter MacCallum Cancer Centre. Male and female mice were used at 6–10 weeks of age. The study was conducted according to the Australian Code of Practice for the care and use of animals for scientific purposes and the Australian National Health and Medical Research guidelines and

Results

We previously made the surprising finding that CD8+ CTLs generated by B6 mice homozygous for the BP allele of GzmB efficiently kill target cells infected with MCMV whereas those of congenic mice homozygous for BW fail to do so. This lack of cytotoxicity in vitro translates into marked susceptibility of mice homozygous for GzmBW to acute infection with MCMV Δ157 in vivo, as 100% of these mice die from overwhelming viral hepatitis and liver failure within 7 days, whereas GzmBP-expressing mice

Discussion

Although they recognise and bind dangerous cells via very different mechanisms, CTL and NK cells share a single, contact-dependent pathway to inflict target cell death once a stable immune synapse has formed. Apart from erythrocytes, which do not express major histocompatibility (MHC) molecules, any somatic cell can be eliminated via ‘granule exocytosis’ – most commonly because they are harbouring a virus or are on a path to malignant transformation [1,2]. Through granule exocytosis, two quite

Conclusion

The current study has advanced our investigations into the mechanism through which heterogeneity in GzmB structure and subtle variations in its enzymatic function can lead to radical differences in outcome following MCMV infection in vivo. Using a variety of biochemical and proteomic approaches, we identified and characterised a number of endogenous and viral proteins which are differentially processed by the GzmBW and GzmBP alloforms. We found no definitive evidence that activation of

Credit author statement

Vivien Sutton – conceptualisation; investigation, supervision of research staff; writing of original manuscript draft; review and editing; Chris Andoniou - conceptualisation; investigation; Michael Leeming – data curation; formal analysis; manuscript review and editing; Colin House – investigation and methodology; data curation; manuscript review and editing; Sally Watt – investigation and methodology; Sandra Verschoor – investigation and methodology; Annette Ciccone – investigation and

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The work described here was supported by Program and Project grants to JAT, IV, MD-E and CA from the National Health and Medical Research Council of Australia. JAT is supported through generous philanthropic support as a Rosie Lew Fellow of the Peter Mac Research Foundation.

References (47)

  • J. Sun et al.

    A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9)

    J. Biol. Chem.

    (1997)
  • C.A. Ray et al.

    Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme

    Cell

    (1992)
  • I. Voskoboinik et al.

    Perforin and granzymes: function, dysfunction and human pathology

    Nat Rev Immunol

    (2015)
  • M. Barry et al.

    Cytotoxic T lymphocytes: all roads lead to death

    Nat Rev Immunol

    (2002)
  • J.A. Lopez et al.

    Rapid and unidirectional perforin pore delivery at the cytotoxic immune synapse

    J. Immunol.

    (2013)
  • R.H. Law et al.

    The structural basis for membrane binding and pore formation by lymphocyte perforin

    Nature

    (2010)
  • V.R. Sutton et al.

    Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation

    J. Exp. Med.

    (2000)
  • D. Kaiserman et al.

    The major human and mouse granzymes are structurally and functionally divergent

    J. Cell Biol.

    (2006)
  • S.P. Cullen et al.

    Human and murine granzyme B exhibit divergent substrate preferences

    J. Cell Biol.

    (2007)
  • N.J. Waterhouse et al.

    A ‘polarized’ look at alpha-tubulin cleavage by granzyme B

    Cell Death Differ.

    (2006)
  • D.A. Anthony et al.

    Functional dissection of the granzyme family: cell death and inflammation

    Immunol. Rev.

    (2010)
  • V.R. Sutton et al.

    Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes

    J. Immunol.

    (1997)
  • K.Y. Thia et al.

    The granzyme B gene is highly polymorphic in wild mice but essentially invariant in common inbred laboratory strains

    Tissue Antigens

    (2007)
  • Cited by (2)

    • Beyond target cell death – Granzyme serine proteases in health and disease

      2022, Molecular Aspects of Medicine
      Citation Excerpt :

      In contrast, granzyme deficient mice have severely impaired MCMV control, although unlike perforin deficient mice, granzyme deficient mice survive infection again highlighting differences in perforin versus granzyme function (van Dommelen et al., 2006). In the case of MCMV, direct cleavage of MCMV proteins by granzymes may be more important for viral control than granzyme-dependent cytotoxicity (Andoniou et al., 2014; Sutton et al., 2020). Ectromelia is one of the few reported viral models where granzyme deficiency can phenocopy perforin loss in terms of mortality during infection (Müllbacher et al., 1999a, 1999b).

    View full text