Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division

Abstract

Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.Several animal DNA viruses, including bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV), maintain their genomes as nuclear extrachromosomal multicopy plasmids in latently infected proliferating host cells. Each of these viruses carries a sequence-specific DNA binding protein which contains the domain required to bind specific receptor complexes on mitotic chromatin (reviewed in references 15, 27, and 43). Viral genomes are tethered to host mitotic chromosomes by segregation proteins at multimeric binding sites. During cell division, the viral genome is partitioned into daughter cells. Therefore, it is widely accepted that segregation protein tethering and multimeric binding to the viral genome are necessary and sufficient to impart a segregation/partitioning function to viral genomes in proliferating cells. However, the identity of the mitotic receptor and how it interacts with the segregation proteins remain elusive.The E2 protein provides a segregation/partitioning function in BPV1 by binding of E2 multimeric binding sites. E2 is a multifunctional sequence-specific DNA binding protein that is required for viral genome replication (10, 62, 67-69), transcriptional activity (60, 65), and viral genome maintenance (2,23,38,51,59). The E2 protein is comprised of an N-terminal transactivation domain (TAD), a flexible hinge region, and a C-terminal DNA binding/dimerization domain (19). BPV1 E2 associates with mitotic chromosomes throughout mitosis, and this interaction is depende...

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…Recently, Silla et al [158] have reported that the formation of the BPV-1 E2 segregation complex is Brd4 independent, which is also the case for all HPVs [159]. Silla et al [158] additionally concluded that the formation of segregation-competent complexes depends on a certain protein level threshold and that multiple E2 molecules are present in a single segregation complex together with cellular proteins, including certain components of the transcription machinery.…”

“…shown that the segregation complex in papillomaviruses contains multiple E2 proteins and also the viral genome should contain at least 5 E2 protein recognition sites [158], it can be expected that a strong chromosomal binding is needed to assure efficient partitioning during cell division. Also, the local organization of chromatin is expected to greatly influence the ability of binding nanoparticles during mitosis.…”

“…Murine leukemia virus (MuLV) (p12) [133,134] Human immunodeficiency virus (HIV) (LEDGF/p75) [141] Avian sarcoma virus (ASV) (IN) [132] Foamy virus (FV) (Gag) [143,144] Epstein Barr virus (EBV) EBNA1 [147][148][149] Kaposi sarcoma-associated herpesvirus (KSHV) LANA [151,152,154] Bovine (BPV) and human papillomavirus (HPV) E2 [155][156][157][158] Simian vacuolating virus 40 (SV40) unknown [161,162] …”

Identification and Analysis of Papillomavirus E2 Protein Binding Sites in the Human Genome

“…Histones were immunoprecipitated with antibodies for H3 (ab1791; Abcam), H3Ac (06-599; Millipore), and H3dimeK4 (07-030; Millipore). Brd4 was immunoprecipitated with 2 g each of rabbit polyclonal antibodies Brd4B and Brd4C (47). DNA was extracted from immunoprecipitated and input material and analyzed in triplicate by quantitative PCR (qPCR) on the 7900HT real-time PCR system (Applied Biosystems).…”

The Role of E2 Proteins in Papillomavirus DNA Replication

“…A recent study using chimeric BPV1 E2 proteins has shown that attachment of the protein to chromatin is not sufficient for proper segregation. Successful partitioning of virus genomes during cell division is determined by effective formation of the segregation-competent complex which does not necessarily involve the Brd4 protein (Silla et al, 2010).…”