The interaction of the gammaherpesvirus 68 orf73 protein with cellular BET proteins affects the activation of cell cycle promoters

Many gammaherpesviruses promote lymphocyte proliferation and can persist in the lymphocyte compartment as well as in a number of other cell types, including endothelial and epithelial cells and fibroblasts (16, 17, 19, 56, 59). To ensure episome maintenance in these dividing cells, gammaherpesviruses express proteins that facilitate the replication of latent viral episomes and ensure the segregation of these viral genomes into progeny cells during cell division. The Epstein-Barr virus (EBV) EBNA-1 protein fulfills these essential functions (49, 62), as do the proteins encoded by open reading frame 73 (ORF73) of the gamma-2 herpesviruses (rhadinoviruses) Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri (HVS), and murine gammaherpesvirus 68 (MHV-68) (4, 20, 28, 29, 43). The orf73 protein of KSHV, latency associated nuclear antigen 1 (LANA-1), is well characterized and has multiple functions. LANA-1 mediates replication and episome maintenance, acts as a transcriptional repressor and activator, and deregulates the cell division cycle (3, 24, 50). Some of these functions are mediated via the interaction of LANA-1 with a number of cellular proteins, including p53 (23), the retinoblastoma protein (48), MeCP2 (35), mSin3 (36), and multiple members of the BET (bromodomain and extra terminal domain) family of proteins (45, 47, 65). The MHV-68 orf73 protein, the KSHV LANA-1 homolog, is critical for the establishment and maintenance of a latent infection in mice (20, 43). MHV-68 orf73 is expressed in latently infected cells as well as during lytic infection (2, 15, 19, 51). The molecular details of how the MHV-68 protein functions are still largely unexplored. BET proteins interact via their bromodomains with acetylated histones (13, 30, 33) and are highly conserved, with members in plants, yeast, Drosophila melanogaster, and up to mammals (18). An additional characteristic feature of BET proteins is the highly conserved extra terminal (ET) domain (see Fig. ​Fig.1),1), which serves as a protein-protein interaction module in Brd2, Brd3, and Brd4 with KSHV LANA-1 (45, 47, 65) and between the yeast (Saccharomyces cerevisiae) BET protein Bdf1 and the TAF7 subunit of the general transcription factor TFIID (39). Mammalian BET proteins are encoded by four genes, Brd2, Brd3, Brd4, and Brd6, out of which Brd2, also called RING3, and Brd4 are the best characterized. Brd2 is a transcriptional regulator that plays a role in cell cycle regulation (11, 12, 33, 55). Brd2 overexpression in B lymphocytes in vivo in mice has been shown to induce lymphoma (26). Brd4 interacts with pTEFb (transcriptional elongation factor b) and thereby promotes RNA polymerase II (Pol II)-dependent transcriptional elongation (7, 32, 61). Brd4 overexpression and depletion both result in deregulated cell cycle progression (14, 38, 45). Recently, Brd4 has been shown to play an important role in the G1/S transition through its ability to stimulate transcription of G1/S-specific genes, among them, cyclin D1 and cyclin D2 (42). Furthermore, the C-terminal domain of the long isoform of Brd4 (Brd4L) (amino acids [aa] 1 to 1362) serves as a chromatin tether for different papillomaviruses via its interaction with their E2 proteins (1, 6, 63, 64). Brd4 is critical for papillomavirus E2 transcriptional activation (31, 40, 54) and may play a role in its transcriptional repression function (53, 60). The same C-terminal domain of Brd4L has recently been shown to interact with the transcriptional elongation factor pTEF and to inhibit human immunodeficiency virus type 1 (HIV-1) Tat-mediated, pTEF-dependent transcription (7). We have shown previously that KSHV LANA-1 interacts with Brd2 and Brd4 via their conserved ET domains (45, 57, 58). This interaction contributes to the association of LANA-1 with cellular heterochromatin and modulates the transcriptional activator role of the short isoform of Brd4 (Brd4S) (aa 1 to 722), which lacks the pTEFb interaction domain and by itself activates the cyclin E promoter (7, 45, 57, 58). FIG. 1. The MHV-68 orf73 protein interacts with the carboxy termini of the cellular BET proteins Brd2/RING3, Brd4S, and Brd3/ORFX. (A) Schematic depiction of the human BET proteins. BR1, bromodomain 1; BR2, bromodomain 2. (B) High degree of sequence conservation ... In this study we show that Brd2, Brd3, and Brd4 also interact with the MHV-68 orf73 protein. By identifying and mutating a binding site for Brd4 and Brd2 in the MHV-68 orf73 protein, we show that the orf73/BET interaction is crucial for the ability to activate the cyclin D1, D2, and E promoters. The results pinpoint the binding site for two BET proteins in a rhadinoviral orf73 protein and indicate that, similarly to papillomavirus E2, a rhadinoviral orf73 protein utilizes a member of the BET protein family to exert its transcriptional activation function.