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Published on: May 15, 2014
Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3
Marcelo Marcet-Palacios1, Brenda Lee Duggan, Irene Shostak
1Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Abstract:
Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.
Insights
Cytotoxic T lymphocytes use Granzyme B to cleave eukaryotic initiation factor 4 gamma 3, halting viral protein synthesis. This mechanism prevents infectious virus production, even when caspases are inhibited.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- Cytotoxic T lymphocytes (CTLs) eliminate virus-infected cells.
- Granzyme B (GrB) induces apoptosis via substrate cleavage.
- Apoptotic cells can still produce infectious viruses, necessitating mechanisms to inhibit viral replication.
Purpose of the Study:
- To investigate a novel role for Granzyme B in inhibiting viral production.
- To identify Granzyme B targets involved in viral protein synthesis.
- To elucidate the mechanism by which GrB suppresses infectious virion formation.
Main Methods:
- Proteomic analysis to identify Granzyme B targets.
- In vitro cleavage assays using Granzyme B and purified eIF4G3.
- Treatment of virus-infected cells with Granzyme B or CTLs.
- Assessment of protein synthesis rates and infectious virion production.
- Inhibition of caspase activity using zVAD-fmk.
Main Results:
- Identified eukaryotic initiation factor 4 gamma 3 (eIF4G3) as a novel Granzyme B target.
- Demonstrated specific cleavage of eIF4G3 by Granzyme B at the IESD(1408)S sequence.
- Observed Granzyme B-mediated degradation of eIF4G3 and reduced translation rates.
- Showed Granzyme B treatment halts viral protein synthesis and decreases infectious vaccinia virus production in infected Jurkat cells.
- Confirmed Granzyme B's inhibition of viral translation is caspase-independent.
Conclusions:
- Granzyme B targets eIF4G3, a key protein synthesis factor, to inhibit viral translation.
- This Granzyme B-mediated mechanism effectively suppresses the production of infectious viruses.
- The findings reveal a novel antiviral strategy employed by CTLs, independent of caspase activation.
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