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Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like
J Bertin1, S M Mendrysa, D J LaCount
1Institute for Molecular Virology, University of Wisconsin-Madison 53706, USA.
Abstract:
Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.
Insights
Baculovirus P35 protein prevents programmed cell death by inhibiting CED-3/ICE-like proteases. Specific mutations show cleavage is necessary but not sufficient for P35
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Baculovirus P35 is a protein inhibitor of CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases.
- P35 prevents programmed cell death (apoptosis) in various organisms.
- Understanding P35's mechanism is key to controlling apoptosis.
Purpose of the Study:
- Identify essential P35 domains for suppressing virus-induced apoptosis in insect cells.
- Determine the role of P35 cleavage in its antiapoptotic activity.
- Investigate the nature of the protease targeted by P35 during baculovirus infection.
Main Methods:
- Site-directed mutagenesis was used to alter the P35 protein sequence.
- In vitro cleavage assays were performed using insect cell extracts and recombinant P35.
- Apoptotic suppression was assessed in insect cells infected with baculovirus.
Main Results:
- A specific cleavage site (DQMD-87G) within P35 is crucial for its antiapoptotic function.
- Mutating the P1 residue (D87A) abolished both P35 cleavage and apoptosis suppression.
- Mutating the P4 residue (D84A) abolished apoptosis suppression but not P35 cleavage, indicating cleavage is insufficient.
- Insect cells undergoing apoptosis possess a CED-3/ICE-like protease activity that cleaves P35.
- Wild-type P35 inhibited this protease activity, while mutated P35 did not.
Conclusions:
- Baculovirus infection activates a novel CED-3/ICE-like protease.
- P35 inhibits this protease through stoichiometric interaction and cleavage.
- P35's broad inhibitory activity against diverse proteases explains its effectiveness in preventing apoptosis.