A naturally occurring C-terminal truncated isoform of the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus does not associate with viral episomal DNA

Abstract

The latency-associated nuclear antigen (LANA) encoded by orf73 of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to viral episomal DNA and nuclear heterochromatin in infected cells. A 3·2 kb transcript in KSHV-positive primary effusion lymphoma (PEL) cells (BCP-1 and BC-3) encoding a C-terminal truncated form of LANA (LANA-Δ76) has been identified. This transcript has the addition of a poly(A) tail at nt 3264 of orf73 resulting in an in-frame stop codon (TAA) effectively truncating LANA by 76 aa (∼8 kDa). Examination of the coding region revealed the presence of a non-canonical polyadenylation signal (AGTAAA) 17 nt upstream of the poly(A) tail. The protein expressed from this transcript is representative of the faster migration of the LANA doublet bands observed by SDS-PAGE and Western blot. Mutation of the poly(A) signal from AGTAAA to TGTACA produced a protein that co-migrated with the larger LANA isoform. A C-terminal LANA-Δ76 EGFP fusion protein localized to the nucleus but did not co-localize with endogenous LANA in BCP-1 cells, or heterochromatin in HEK293 cells. Using an electrophoretic mobility shift assay (EMSA), the authors were able to show that LANA-Δ76 does not bind to the KSHV terminal repeat motif known to interact with LANA. These data provide evidence for the presence of an isoform of LANA that may perform alternative functions in KSHV-infected cells.