Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: Formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AI DS patients are universally associated with infection by the presumed causa tive agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 ( Latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion Lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSH V has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol ace tate-induced PEL cell lines can induce cultured primary human dermal microv ascular endothelial cells (DMVEC) to form colonies of proliferating latentl y infected spindle-shaped cells, all of which express the KSHV-encoded LANA 1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-l ike phenotype of adjacent uninfected DMVEC, Each infected culture could als o be expanded into a completely spindloid persistently infected culture dis playing aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phos phonoacetic acid or gangciclovir, but these antiherpesvirus agents had litt le effect on the propagation of already latently infected spindloid culture s. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle p roteins, and 1 to 2% of the cells also formed typical herpesvirus DNA repli cation compartments, displayed cytopathic rounding effects, and expressed l ate viral antigens, We conclude that de novo KSHV infection induces a spind le cell conversion phenotype in primary DMVEC cultures that is directly ass ociated with latent state expression of the LANA1 protein. However, these c ultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV v irion preparations.