Kaposi's sarcoma-associated herpesvirus can productively infect primary human keratinocytes and alter their growth properties

Previous studies have shown the presence of Kaposi's sarcoma-associated her pesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basa l layer of the epidermis overlying plaque-stage nodular lesions of cutaneou s Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands with in KS lesions. We infected primary cell cultures of human keratinocytes wit h KSHV/HHV8, At 6 days post infection, transcription of viral genes was det ected by reverse transcriptase PCR (RT-PCR), and protein expression was doc umented by an immunofluorescence assay with an anti-LANA monoclonal antibod y. To determine whether the viral lytic cycle was inducible by chemical tre atment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecano ylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcri ption of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary hu man cells (human umbilical vein endothelial cells), were infected with conc entrated supernatant of KSHV-infected, TPA-induced keratinocytes and the pr esence of viral transcripts was confirmed by RT-PCR The uninfected keratino cytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infec ted 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 sti ll 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, lik e other herpesviruses, can infect and replicate in epithelial cells in vitr o and suggest that in vivo these cells may play a significant role in the e stablishment of KSHV/HHV8 infection and viral transmission.