Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus

doi:10.1128/JVI.74.6.2867-2875.2000

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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 subfamilyand encodes ~80 open reading frames (ORFs). Among them are a fewcandidates for immediate-early genes (e.g., K5). We developeda monoclonal antibody (MAb), 328C7, against the K5 antigen. ThisMAb reacted with the K5 gene product by immunoscreening of a cDNAlibrary from BCBL-1 cells, and this result was confirmed by transfectionof the K5 ORF into Cos-7 cells. After induction of lytic infectionby treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cellsas early as after 4 h of induction. Immunoelectron microscopyshowed that the K5 antigen was situated mainly in the endoplasmicreticulum but was not present on the virion or in the nucleus.Northern blotting with a K5-specific probe revealed a single transcriptof 1.2 kb, while Western blotting showed the antigen to be a 36-kDapolypeptide. The 5' and 3' ends were then determined by rapidamplification of cDNA, followed by sequencing of RACE products,and a splice was revealed upstream of the K5 ORF. K5 expressionwas unaffected by the respective DNA and protein synthesis inhibitorsphosphonoformic acid and cycloheximide plus actinomycin D, confirmingits immediate-early nature. Transient-transfection assays showedthat the K5 promoter was transactivated by ORF 50 (KSHV Rta),a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibitedno transregulation of its own promoter or those of DNA polymeraseand 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 furtherinvestigation.

^* 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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