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. 1998 Jun;72(6):4980-8.
doi: 10.1128/JVI.72.6.4980-4988.1998.

Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene

S Muralidhar  1 , A M Pumfery, M Hassani, M R Sadaie, M Kishishita, J N Brady, J Doniger, P Medveczky, L J Rosenthal
Affiliations

Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene

S Muralidhar et al. J Virol. 1998 Jun.

Erratum in

  • J Virol 1999 Mar;73(3):2568

Abstract

The recently identified human herpesvirus 8 (HHV-8, or Kaposi's sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi's sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865-1869, 1994; P. S. Moore et al., J. Virol. 70:549-558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0. 7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641-6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715-719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342-346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.

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Figures

FIG. 1
FIG. 1
Map of HHV-8 LUR showing the locations of the conserved and unique ORFs, including sequences tested for transforming ability, and a schematic diagram of the ∼140-kb LUR sequence flanked by terminal repeats (TR). HHV-8 fragments tested in the focus-forming assay (17, 23, and 199) are shown as solid bars below the map. The locations of T0.7 and kaposin (K12) sequences within fragment 23 are indicated. The solid arrows indicate HHV-8 ORFs conserved in HVS, and the open arrows indicate the unique HHV-8 ORFs. Repeat regions are shown as small filled rectangles above the ORFs (frnk, vnct, waka/jwka, zppa, moi, and mdsk). KS330 and KS631 are the fragments of HHV-8 that were identified first. CBP, complement binding protein; ss DBP, single-stranded DNA binding protein; gB, glycoprotein B; DNA pol, DNA polymerase; DHFR, dihydrofolate reductase; TS, thymidylate synthetase; MIP, macrophage inflammatory protein; nut-1, nuclear transcript 1; Teg, tegument protein; TK, thymidine kinase; gH, glycoprotein H; MCP, major capsid protein; gM, glycoprotein M; UDG, uracil DNA glucosidase; gL, glycoprotein L; R-trans, transactivator; gX, glycoprotein X; vIRF, viral interferon regulatory factor; RRS, ribonucleotide reductase, small; RRL, ribonucleotide reductase, large; CycD, cyclin D homolog; Adh, immunoglobulin family adhesion protein; GCR, G-protein-coupled receptor. The map was adapted from Fig. 1 of reference with the permission of the publisher.
FIG. 2
FIG. 2
Kaposin-transformed Rat-3 cells induced high-grade undifferentiated sarcomas in athymic nu/nu mice. (A) Typical focus induced in pBK/kap-transfected Rat-3 cells. The focus of refractile randomly oriented multilayered transformed cells is shown against a background of parental Rat-3 cells (magnification, ×60). (B) Tumor development in an athymic nu/nu mouse at 2 weeks postinoculation with kaposin-transformed Rat-3 cells. (C) Representative section of a tumor induced by kaposin-transformed Rat-3 cells, fixed with formalin and stained with hematoxylin and eosin. Magnification, ×200.
FIG. 3
FIG. 3
Expression of kaposin mRNA in tumor-derived cell lines. Total RNA was extracted from Rat-3, R3/kap-TL1, and R3/kap-TL2 cells. The RNA was separated on a 1% formaldehyde-agarose gel, blotted onto a Zetabind nylon membrane, and probed under stringent conditions with 32P-labeled kaposin DNA. The 1.1-kb unspliced and 0.8-kb spliced messages for kaposin are shown. Positions of 28S (4.7-kb) and 18S (1.9-kb) rRNA markers are indicated on the left.
FIG. 4
FIG. 4
Kyte-Doolittle hydrophobicity plot of kaposin showing the predominant hydrophobic domains and locations of the hydrophilic kaposin peptides used to generate the antikaposin antibodies. kap-1 and kap-2 are hydrophilic peptide sequences used to generate anti-kap-1 and anti-kap-2 antibodies, respectively.
FIG. 5
FIG. 5
Anti-kap-2 antibody detected bacterially expressed kaposin fusion protein. E. coli BL21(DE3)(pLysS), transformed by either pET30b-kap or pET30b, was induced for 90 min with 1 mM IPTG (I) or uninduced (U). Proteins were extracted in SDS-gel dye buffer, separated by SDS-PAGE, and electroblotted onto Immobilon-P membranes. (A) Western blot showing the 17 kDa S-Tag/kaposin fusion protein in the pET30b-kap (lane I), using the S-Tag/alkaline phosphatase conjugate (Novagen). (B) Western blot showing the S-Tag/kaposin fusion protein seen in panel A detected with anti-kap-2 antibody. Sizes on the left are indicated in kilodaltons.
FIG. 6
FIG. 6
Detection and localization of kaposin protein in transformed Rat-3 cells, tumor-derived cells, and the PEL cell line BCBL-1. IFA shows cytoplasmic staining of kaposin protein in tumor-derived cell line R3/kap-TL1, using anti-kap-2 antibody (1:50) (panel 1A). Also shown are R3/kap-TL1 cells stained with preimmune serum (1:50) (panel 2A) and vector-transfected cell line R3/BK-G1 stained with anti-kap-2 antibody (1:50) (panel 3A). BCBL-1 cells stained with anti-kap-2 antibody (1:5) and preimmune serum (1:5) are shown in panels 4A and 5A, respectively. Intense staining with anti-kap-2 antibody in a restricted region of the cytoplasm of R3/kap-TL1 and BCBL-1 cells are indicated by arrows in panels 1A and 4A, respectively. DAPI staining of nuclei of the above-specified fields are shown in panels 1B, 2B, 3B, 4B, and 5B, respectively. Magnifications: panels 1 to 3 (A and B), ×1,800; panels 4 and 5 (A and B), ×3,000.
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