Prokaryotic expression of an immediate-early gene of human herpesvirus 6 and analysis of its viral antigen expression in human cells.
Segments of an immediate-early (1E) protein (1E03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as beta-galactosidase fusion proteins in Escherichia coli. Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M). The fusion protein expressed in E. coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody. Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test. Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A. The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D. Further, it was also detected as early as 4 h after infection.
Takeda
K
K
Department of Virology, Osaka University, Japan.
Nakagawa
N
N
Yamamoto
T
T
Inagi
R
R
Kawanishi
K
K
Isegawa
Y
Y
Yamanishi
K
K
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005077:Exanthema Subitum / Q000097:blood / Q000276:immunology / Q000821:virology; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D011387:Prokaryotic Cells / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins
D000914:Antibodies, Viral; D000956:Antigens, Viral; C103842:IE03 protein, herpes simplex virus 6; D017874:Immediate-Early Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C072628:herpes simplex virus IE3 protein, Human herpesvirus 1; D003609:Dactinomycin; D003513:Cycloheximide