Discrete cleavage patterns of pseudorabies virus immediate early protein (IE180) seen in some cell lines upon extraction after cycloheximide reversal.
Pseudorabies virus (PrV) encodes for a single and essential immediate early phosphoprotein designated IE180. In this study, IE180 was examined in lysates from various cell lines infected at high multiplicities under cycloheximide inhibition of protein synthesis and subsequent reversal. Three distinct protein patterns of IE180 which were cell-specific and dependant on the extraction procedure were revealed. Detergent lysates of PrV infected MDBK cells yielded almost exclusively wild type IE molecule (180 kDa). In contrast, SSG/94 cells, VERO or CV-1 cells did not yield 180 kDa molecules but predominantly a shorter variant of approximately 60 kDa in molecular mass. Additional bands of about 50/55 kDa were also detected in lysates of SSG/94 and VERO cells by immunoprecipitation. Lysates of CV-1 and MDBK cells also yielded a 120 kDa molecule. The smaller molecular mass bands occurred in the presence of PMSF and aprotinin however, cleavage was blocked completely by addition of N alpha-p-Tosyl-L-lysine chloromethyl ketone (TLCK) into the lysis buffer. Moreover, an ability of the shorter IE180 variants to bind heparin was also revealed in the study. These data provide useful insights on protease profiles encountered among different PrV susceptible cells and indicates the use of appropriate protease inhibitors such as TLCK to protect IE180 under these experimental conditions.
Garmendia
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Department of Pathobiology, University of Connecticut, Storrs 06268, USA. Garmendi@UCONNVM.UCONN.EDU
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D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D011558:Herpesvirus 1, Suid / Q000187:drug effects / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013552:Swine; D014709:Vero Cells
D000914:Antibodies, Viral; D000956:Antigens, Viral; D017874:Immediate-Early Proteins; D011500:Protein Synthesis Inhibitors; C047583:pseudorabies virus immediate early proteins; D006493:Heparin; D003513:Cycloheximide; D010450:Endopeptidases