Comparison of the Immediate Early Polypeptides of Human Cytomegalovirus Isolates

Abstract

421 SUMMARYThe in vivo and in vitro synthesized immediate early (IE) polypeptides of three isolates of human cytomegalovirus (CMV) have been compared by SDSpolyacrylamide gel electrophoresis. Of the 11 IE polypeptides detected early in infection, one was very abundant and had a different apparent mol. wt. depending on the isolate. This difference may be useful in the classification of CMV into strains.There is growing awareness of the importance of cytomegalovirus (CMV) as a human pathogen. As well as producing a variety of syndromes such as cytomegalic inclusion disease, interuterine death, congenital defects and interstitial pneumonia in transplant patients (Weller, 1971), CMV can be maintained in the latent state (Diosi et aL, 1969). Furthermore, transformation studies with hamster embryo fibroblasts (Albrecht & Rapp, 1973) and human embryo fibroblasts (Geder et al., 1976) have suggested that CMV may have oncogenic potential.Although there exist many isolates of CMV, it is not known how these relate to each other. It is unclear to what extent CMV isolates can be classified into different strains, if distinct strains are always isolated from the same clinical source, or whether all isolates are capable of producing the range of syndromes associated with CMV. Studies on the antigenic relatedness of CMV isolates using complement fixation and cross-neutralization tests have not been conclusive (Michelson-Fiske, 1977;Waner & Weller, 1978). DNA-DNA renaturation kinetics have shown a high degree of genetic homology (80 %) between different CMV isolates, and although studies of CMV DNA with restriction endonucleases have demonstrated some differences between isolates, these do not fall into patterns which could be used to group these isolates (Kilpatrick et aL, 1976). Another approach has been to analyse the polypeptides of the purified virion by SDS-polyacrylamide gel electrophoresis (Gupta et al., 1977), but the four strains studied (AD169, C87, Burke and Towne) showed great similarities even between their major capsid proteins. This paper reports a difference between three laboratory strains of CMV which emerged during a study of the immediate early (IE) polypeptides of several isolates of CMV, and which may be useful as a diagnostic tool.The cells used in the experiments were the Flow 2002 line of human embryo lung fibroblasts and the viruses were the CMV isolates AD169, Davis and Towne (see Kilpatrick et al., 1976 for their origin). For the in vivo studies, monolayers of cells in 30 mm diam. dishes were infected with virus at a m.o.i, of 5 in the presence of 50 ag/ml cycloheximide (CH) (Sigma) to inhibit protein synthesis. Uninfected controls were treated with 0.2 ml phosphate-buffered saline (PBS). After adsorption at 37 °C for 1 h, the monolayers were washed three times and then overlaid with Eagle's medium supplemented with-10 % foetal calf serum containing 50 #g/ml CH. At 12 h post-infection (p.i.), 5/~g/ml actinomycin D (Act D) (Sigma) was added to prevent further transcription, and 15 min later the plates ...

Human Cytomegalovirus Infections in vitro after Treatment with Arildone

“…The apparent molecular weight of the predominant IE protein of AD169 virus determined by S D S -P A G E was 74000 and 70000 for the less abundant satellite protein. Strain differences in the electrophoretic mobility of the predominant IE protein (Cameron & Preston, 1981 ;Gibson, 1981Gibson, , 1983Stevens et al, 1984) are supportive evidence of the viral nature of this protein and this point is illustrated in Fig. 4(a) by comparison of AD169 virus and prototype virus Kerr.…”

“…Using this protocol, up to 11 IE proteins have been identified in HCMV-infected cells including the predominant IE protein, which is readily resolved after cycloheximide enhancement (Stinski, 1978 ;Cameron & Preston, 1981 ;Gibson, 1981). In the present study, expression of IE proteins of H C M V and the predominant IE protein in particular was used as a measure of early transcription and translation after virus infection.…”

Human Cytomegalovirus: Recent Aspects from Molecular Biology

“…The protein accumulates primarily in the nucleus of the cell (Michelson-Fiske et al, 1977). The Mr of the predominant IE protein, which varied slightly between different strains of HCMV, was estimated to be 75K by SDS-PAGE (Blanton & Tevethia, 1981 ;Cameron & Preston, 1981 ;Gibson, 1981 b;Wilkinson et al, 1984). This value differs considerably from that calculated from the sequence.…”

Resistance to Methylation de novo of the Human Cytomegalovirus Immediate Early Enhancer in a Model for Virus Latency and Reactivation in vitro

“…Viral mRNAs synthesized in the presence of inhibitors of protein synthesis and the proteins they encode are defined as IE mRNAs and immediate early antigens (lEAs), respectively. At least four lEAs can be detected in infected cells by immunoprecipitation, the predominant (major) lEA being a phosphorylated protein showing strain heterogeneity in apparent molecular weight (Blanton & Tevethia, 1981;Cameron & Preston, 1981;Gibson, 1981 ;Michelson et al, 1979;Stinski, 1978;Tanaka et al, 1979). Within a few hours of infection of either permissive or non-permissive (e.g.…”