ELSEVIER Journal of Virological Methods 64 (1997) 171-179 Discrete cleavage patterns of pseudorabies virus immediate early Journal of Virological Methods protein (IE180) seen in some cell lines upon extraction after cycloheximide reversal Antonio E. Garmendiaa,*, Zhiqiang Lub, Edan R. Tulman” “ Department of Pathobiology, Uniaersity of Connecticut, Stows, CT 06268, USA ‘Plum Island Animal Disease Center, USDA, ARS, Greenport, NY 11944, USA Accepted 4 November 1996 Abstract 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 SO/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 Na-p-Tosyl-L-lysine chloromethyl ketone (TLCK) into the lysis buffer. Moreover, an ability of the shorter IEl80 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. 0 1997 Elsevier Science B.V. Keywords: Pseudorabies; Immediate early protein; Cycloheximide Abbreriations: SSG/94, a cell line derived from skin explants of “‘cSLA inbred swine; SAPA, Staphylococcus aureus protein A ; PAGE-SDS, polyacrylamide gel electrophoresis with sodium dodecyl sulphate; PrV, pseudorabies virus; IE180, immediate early protein 180; CHX, cycloheximide. * Corresponding author. Tel.: + 1 203 4860821; fax: + 1 203 4862794; e-mail: Garmendi@UCONNVM.UCONN.EDU 1. Introduction The genetic pool of herpesviruses in general, consists of essential and non essential genes which code either for structural or non-structural viral 0166.0934/‘97,!$17.00 0 1997 Elsevier Science B.V. All rights reserved PI1 SO1 66-0934(96)02 157-X