Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus.

Abstract

Over 85% of patients with Kaposi's sarcoma (KS) are seropositive for antibodies to the latency-associated nuclear antigen (LANA) expressed in B cell lines infected with Kaposi's sarcoma-associated herpesvirus (KSHV).The presence of antibodies to LANA strongly correlates with the risk of developing the disease.However, the identity of the protein(s) comprising LANA and the corresponding gene(s) has remained unclear.To identify potential latent gene candidates for LANA, we probed total RNA extracted from BCBL-1 cells (a B cell line latently infected with KSHV) using lambda clones that span the KSHV genome.One region encoding latent transcripts spanned KSHV open reading frames (orfs) 71 (K13), 72 (v-cyclin), and 73.Among these, however, only orf 73, when expressed in heterologous mammalian cell systems, reacted with KSHV antibody-positive human sera, resulting in a punctate nuclear staining pattern reminiscent of LANA in BCBL-1 cells.Furthermore, extracts from cells expressing the orf 73 protein product specifically blocked the binding of KS patient antibodies to LANA.Finally, seroreactivity with recombinant orf 73 protein exactly paralleled reactivity with classical LANA as expressed in BCBL-1 cells, both in KS patients and in other groups.Together, these data support the identification of KSHV orf 73 as the gene encoding the dominant immunogenic component of LANA.( J. Clin.Invest.1997.100:2606-2610.)Key words: HHV8 • KSHV • Kaposi's sarcoma MethodsCell culture.COS7, 293 and CV-1 cells were cultured in DME-H16 supplemented with 10% fetal calf serum (Lifetechnologies, Inc., Gaithersburg, MD), 50 g/ml penicillin and 50 g/ml streptomycin.Cells were cultured at 37 Њ C in 5% CO 2 .BCBL-1 cells were cultured