ER-localization and functional expression of the HBV transactivator MHBst.
In two recently reported cases, integrated hepatitis B virus (HBV) DNAs cloned from hepatocellular carcinoma were found to express a transcriptional transactivator from 3'-terminally truncated HBV surface (preS/S) genes. In this study, we characterized the transactivator at the protein level. Expression of a 3'-truncated preS2/S gene in Spodoptera frugiperda (Sf9) insect cells resulted in a C-terminally truncated middle surface protein of 76 amino acids (MHBst76), which was found to be associated with membranes of the endoplasmic reticulum and retained from Golgi processing and secretion. Accordingly, the microsome fraction of MHBst76-expressing Sf9 cells displayed transactivator activity after electric field-mediated transfer into Chang liver cells. In contrast to full-length MHBs, MHBst76 is unglycosylated, and glycosylation is not required for transactivation as shown by mutation of the glycosylation site at asparagine-4. Since highly purified MHBst76 derived from an E. coli expression system also showed transactivator activity, it is concluded that unglycosylated MHBst76 protein is the authentic transactivating factor. As the transactivator protein derives from inactive MHbs by rearrangements of integrated HBV DNA, it may be important for HBV-associated liver carcinogenesis.
Hildt
E
E
Department of Virus Research, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Urban
S
S
Lauer
U
U
Hofschneider
P H
PH
Kekulé
A S
AS
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D015321:Gene Rearrangement; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006515:Hepatitis B virus / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009036:Moths; D015533:Transcriptional Activation / Q000502:physiology
D000596:Amino Acids; D004279:DNA, Viral; D008562:Membrane Glycoproteins; D001216:Asparagine