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Journal of Virology, March 2001, p. 2435-2443, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2435-2443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

Francesca Cerimele,1,2 Francesca Curreli,1 Scott Ely,3 Alvin E. Friedman-Kien,1,4 Ethel Cesarman,3 and Ornella Flore1,*

Departments of Microbiology1 and Dermatology,4 New York University School of Medicine, New York, New York 10016; Department of Pathology, Weill Medical College of Cornell University, New York, New York 100213; and Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Rome, Italy2

Received 30 June 2000/Accepted 15 November 2000

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission.

* Corresponding author. Mailing address: Department of Microbiology, NYU School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-5313. Fax: (212) 263-7933. E-mail: ornella.flore{at}med.nyu.edu.

Journal of Virology, March 2001, p. 2435-2443, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2435-2443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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