Doctor of Philosophy
Holmes, Shan, Human cytomegalovirus induced plasma membrane glycoproteins in fibroblasts, Doctor of Philosophy thesis, , University of Wollongong, 1990. http://ro.uow.edu.au/theses/2096
The relationship of novel HCMV induced plasma membrane glycoproteins of M.W. 54, 62 and 94 kDa to proteins of the virion envelope and normal cell plasma membranes was investigated. Plasma membrane preparations were isolated from metabolically labelled HCMV infected and uninfected fibroblasts by the FMA and two phase polymer systems procedures, and analysed by SDS PAGE, fluorography and image subtraction analysis, for the initial characterization of HCMV induced proteins of M.W. 26,32-34,38,48,54- 55,62,68,72-74,86,92-94,105,130 and 150 kDa.
The reactivity of antisera raised against virus induced plasma membrane proteins 62 and 94 kDa with 125 I labelled virions and detergent extracted and osmotically fragile virion envelope proteins was assessed by immunoprecipitaion analysis, reducing SDS PAGE and fluorography. All three antisera were reactive with at least one protein of M.W. 54-58 kDa and anti- 54 kDa and anti- 62 kDa sera, were shown to be specfic by immunoaffinity absorption chromatography for reduced HCMV induced novel plasma membrane proteins of 54 and 62 kDa respectively. Both these antisera also neutralized virus in vitro in the absense of complement. All three antisera were also shown to react with the nuclear and plasma membranes of infected fibroblasts by indirect immunoperoxidase immunobinding assays.
Rabbit antisera raised against HCMV induced plasma membrane proteins of 54, 62 and 94 kDa, guinea pig monospecific polyclonal antisera reactive with the gB virion envelope proteins 55/130 kDa, and monoclonal antibodies specific for the virion gB complex, 15D8 (Rasmussen et al., 1985), C H 28-2 (Pereira et al., 1984), 7-17 (Britt et al., 1985) and α 52 (Farrar and Greenaway, 1986) were used to assess the antigenic association of virion envelope proteins and HCMV induced and normal plasma membrane proteins.
Immunoblot and immnoprecipitation analyses of HCMV induced plasma membrane proteins with anti- 54, 62 and 94 kDa sera indicated that HCMV induced plasma membrane proteins of 54, 62 and 94 kDa were antigenically related and reacted with HMWC'S of 130-150 and 500kDa in plasma membrane preparations from infected fibroblasts. Anti-54 kDa sera was shown to be virus specific wheras anti-62 and anti-kDa sera demonstrated a low affinity for host cell proteins although a HMWC of 140 could possibly be virus specific.
HMWC's of 130-150 kDa, also reported for the virion gB glycoprotein complex, were detected when HCMV induced plasma membrane proteins were immunoprecipitated with monospecific polyclonal sera 4PP and 6PP and Mab 15D8 and analysed under non reducing gel conditions. Serum 4PP and Mab 15D8 were highly virus specific whereas serum 6PP cross reacted with low affinity to host cell proteins. Plasma membrane preparations of HCMV infected and uninfected fibroblasts immunnoprecipitat ed with Mabs 15D8, 7-17 , CH28-2 and analysed by reducing SDS PAGE and fluorography showed that proteins of similar M.W. to proteins of the gB glycoprotein complex of virion envelope, namely 130, 94, 68 and 55 kDa were also present in the plasma membranes of infected cells. However HMWC's of 130-150 kDa and reduced proteins in the molecular weight region of 130, 68-70, 55 and 28 kDa were also detected in cell plasma membrane proteins and in plasma membrane preparations isolated at 12h 48h p.i. with Mab 15D8 when the autoradiographs were exposed for an extended time, indicating that normal cell proteins cross react with virion envelope proteins that virus infectivity in vitro.