Role of the C-terminal RDEL motif of the myxoma virus M-T4 protein in terms of apoptosis regulation and viral pathogenesis.
The purpose of this study was to investigate the significance of the C-terminal RDEL motif of the myxoma virus M-T4 protein in terms of apoptosis regulation and role in viral virulence. To accomplish this, a recombinant myxoma virus was created in which the C-terminal RDEL motif of M-T4 was deleted and a selectable marker (Ecogpt) was inserted immediately downstream. We hypothesized that removal of the RDEL motif from M-T4 would alter the subcellular localization of the protein and provide insight into its antiapoptotic role. Surprisingly, removal of the RDEL motif from M-T4 did not affect localization of the protein within the endoplasmic reticulum (ER), but it did reduce the stability of the mutant protein. Pulse-chase immunoprecipitation and endoglycosidase H analysis coupled with confocal fluorescent light microscopy demonstrated that the M-T4 RDEL(-) mutant protein is retained in the ER like wildtype M-T4 and suggests that the C-terminal RDEL motif is not the sole determinant for M-T4 localization to the ER. Infection of cultured rabbit lymphocytes with the M-T4 RDEL(-) mutant virus results in an intermediate apoptosis phenotype compared with the wildtype and M-T4 knockout mutant viruses. A novel myxomatosis phenotype was observed in European rabbits when infected with the recombinant M-T4 RDEL(-) mutant virus. Rabbits infected with the M-T4 RDEL(-) virus on day 9 postinfection exhibited an exacerbated edematous and inflammatory response at secondary sites of infections, particularly the ears. Our results indicate that the C-terminal RDEL motif may not be solely responsible for retention of M-T4 to the ER and that M-T4 may have a dual function in protecting infected lymphocytes from apoptosis and in modulating the inflammatory response to virus infection.
Hnatiuk
S
S
Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, N6G 2V4, Canada.
Barry
M
M
Zeng
W
W
Liu
L
L
Lucas
A
A
Percy
D
D
McFadden
G
G
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000821:virology; D002460:Cell Line; D004431:Ear, External / Q000473:pathology / Q000821:virology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000821:virology; D005455:Fluorescent Antibody Technique; D006207:Half-Life; D006596:Hexosaminidases / Q000378:metabolism; D007249:Inflammation / Q000473:pathology / Q000821:virology; D009233:Myxoma virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity* / Q000502:physiology; D009234:Myxomatosis, Infectious / Q000473:pathology / Q000821:virology; D011233:Precipitin Tests; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
C110150:M-T4 protein, myxoma virus; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014764:Viral Proteins; D006596:Hexosaminidases