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Journal of Virology, March 2000, p. 2867-2875, Vol. 74, No. 6
Department of Microbiology1 and
Department of Cell Biology and
Neurosciences,2 Osaka University Medical School
C1, 2-2 Yamadaoka, Suita 565-0871, and Department of
Microbiology, Hyogo Medical College, Nishinomiya 663-8131,
Hyogo,3 Japan
Received 13 September 1999/Accepted 2 December 1999
Kaposi's sarcoma-associated herpesvirus (KSHV), or human
herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and
encodes ~80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a
monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb
reacted with the K5 gene product by immunoscreening of a cDNA library
from BCBL-1 cells, and this result was confirmed by transfection of the
K5 ORF into Cos-7 cells. After induction of lytic infection by
treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb
328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern
blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5' and 3' ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a
splice was revealed upstream of the K5 ORF. K5 expression was
unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that
the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of
Epstein-Barr virus Rta, but the K5 gene product exhibited no
transregulation of its own promoter or those of DNA polymerase and the
human immunodeficiency virus type 1 long terminal repeat. This is the
first such analysis of an immediate-early gene product; determination
of its specific biological function requires further investigation.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification and Analysis of the K5 Gene of
Kaposi's Sarcoma-Associated Herpesvirus
*
Corresponding author. Mailing address: Department of
Microbiology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3321. Fax: 81-6-6879-3329. E-mail: yamanisi{at}micro.med.osaka-u.ac.jp.
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