Binding of FADD and caspase-8 to molluscum contagiosum virus MC159 v-FLIP is not sufficient for its antiapoptotic function.
Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and alpha2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the alpha6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, alpha2, and alpha6) are critical for the protein's ability to interact with cellular proteins and to block apoptosis.
Garvey
Tara L
TL
Medical Virology Section, Laboratory of Clinical Investigation, Molecular Development of the Immune System Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
Bertin
John
J
Siegel
Richard M
RM
Wang
G H
GH
Lenardo
Michael J
MJ
Cohen
Jeffrey I
JI
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D053200:Fas-Associated Death Domain Protein; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000494:pharmacology; D008969:Molecular Sequence Data; D008977:Molluscum contagiosum virus / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases