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Journal of Virology, June 2001, p. 5614-5626, Vol. 75, No. 12
Molecular Virology Program, Department of
Oncology, The Johns Hopkins University School of Medicine Cancer
Center, Baltimore, Maryland 21231
Received 20 November 2000/Accepted 20 March 2001
Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS
and non-AIDS patients are universally associated with infection by the
presumed causative 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 KSHV has so far eluded
investigators. Here, we demonstrate that infection with supernatant
virions derived from three different tetradecanoyl phorbol
acetate-induced PEL cell lines can induce cultured primary human dermal
microvascular endothelial cells (DMVEC) to form colonies of
proliferating latently infected spindle-shaped cells, all of which
express the KSHV-encoded LANA1 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-like
phenotype of adjacent uninfected DMVEC. Each infected culture could
also be expanded into a completely spindloid persistently infected
culture displaying aggregated swirls of spindle cells resembling those
in KS lesions. Formation of new colonies and plaques was inhibited in
the presence of phosphonoacetic acid or gangciclovir, but these
antiherpesvirus agents had little effect on the propagation of already
latently infected spindloid cultures. In persistently infected
secondary cultures, patches of up to 10% of the spindloid cells
constitutively expressed several early viral lytic cycle proteins, and
1 to 2% of the cells also formed typical herpesvirus DNA replication
compartments, displayed cytopathic rounding effects, and expressed late
viral antigens. We conclude that de novo KSHV infection
induces a spindle cell conversion phenotype in primary DMVEC cultures
that is directly associated with latent state expression of the LANA1
protein. However, these cultures 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 virion preparations.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5614-5626.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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
*
Corresponding author. Mailing address: Molecular
Virology Laboratories, Rm. 3M-08 Bunting-Blaustein Cancer Research
Building, The Johns Hopkins University School of Medicine, 1650 Orleans St., Baltimore, MD 21231. Phone: (410) 955-8684. Fax: (410) 955-8685. E-mail: ghayward{at}jhmi.edu.
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