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Journal of Virology, March 2000, p. 2867-2875, Vol. 74, No. 6
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

Muzammel Haque,1 Jiguo Chen,1 Keiji Ueda,1 Yasuko Mori,1 Kazusi Nakano,1 Yuko Hirata,1 Shiro Kanamori,2 Yasuo Uchiyama,2 Reiko Inagi,1 Toshiomi Okuno,3 and Koichi Yamanishi1,*

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.

* 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.

Journal of Virology, March 2000, p. 2867-2875, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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