Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that acts on viral terminal repeat (TR) DNA to mediate KSHV episome persistence. The two essential components of episome persistence are DNA replication prior to cell division and episome segregation to daughter nuclei. These functions are located within N- and C-terminal regions of LANA. N- and C-terminal regions of LANA are sufficient for TR DNA replication. In addition, N- and C-terminal regions of LANA tether episomes to mitotic chromosomes to segregate episomes to progeny cell nuclei. To generate a tethering mechanism, N-terminal LANA binds histones H2A/H2B to attach to mitotic chromosomes, and C-terminal LANA binds TR DNA and also associates with mitotic chromosomes. Here, we test the importance of the internal LANA sequence for episome persistence. We generated LANA mutants that contain N- and C-terminal regions of LANA but have most of the internal sequence deleted. As expected, the LANA mutants bound mitotic chromosomes in a wild-type pattern and also bound TR DNA as assayed by electrophoretic mobility shift assays (EMSA). The mutants mediated TR DNA replication, although with reduced efficiency compared with LANA. Despite the ability to replicate DNA and exert the chromosome and DNA binding functions necessary for segregating episomes to daughter nuclei, the mutants were highly deficient for the ability to mediate both short- and long-term episome persistence. These data indicate that internal LANA sequence exerts a critical effect on its ability to maintain episomes, possibly through effects on TR DNA replication.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV), termed human herpesvirus 8, is a gamma-2-herpesvirus that is tightly associated with Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease [10, 11, 49, 63]
Efficient segregation is achieved by latency-associated nuclear antigen (LANA)-mediated tethering of the KSHV episome to the host chromosome; mitotic chromosome association is an essential process for KSHV episome maintenance
Since C-terminal LANA binding to KSHV terminal repeat (TR) DNA is essential for episome persistence, we investigated the ability of LANA mutants to bind TR DNA in electrophoretic mobility shift assays (EMSA)
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV), termed human herpesvirus 8, is a gamma-2-herpesvirus that is tightly associated with Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease [10, 11, 49, 63]. LANA permits episome persistence by mediating the replication of, and tethering of the KSHV terminal repeats (TRs) to, host cell chromosomes. Both the N- and C-terminal regions of LANA are essential for episome maintenance. The C-terminal region of LANA ( termed C-terminal LANA) binds to two adjacent sites in each TR, and this binding is essential for TR DNA replication and episome persistence [4, 13, 16, 21, 22, 24, 26, 33, 45]. The internal LANA domains exert an important role in episome maintenance
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