DISCRETE CLEAVAGE PATTERNS OF PSEUDORABIES VIRUS IMMEDIATE-EARLY PROTEIN (IE18O) 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 examin ed in lysates from various cell lines infected at high multiplicities under cycloheximide inhibition of protein synthesis and subsequent rev ersal. Three distinct protein patterns of IE180 which were cell-specif ic and dependant on the extraction procedure were revealed. Detergent lysates of PrV infected MDBK cells yielded almost exclusively wild typ e 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 o f 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 immu noprecipitation. Lysates of CV-1 and MDBK cells also yielded a 120 kDa molecule. The smaller molecular mass bands occurred in the presence o f PMSF and aprotinin however, cleavage was blocked completely by addit ion of N alpha-p-Tosyl-L-lysine chloromethyl ketone (TLCK) into the ly sis buffer. Moreover, an ability of the shorter IE180 variants to bind heparin was also revealed in the study. These data provide useful ins ights on protease profiles encountered among different PrV susceptible cells and indicates the use of appropriate protease inhibitors such a s TLCK to protect IE180 under these experimental conditions. (C) 1997 Elsevier Science B.V.