CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins

Abstract

Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the Nterminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.T he study of papillomaviruses has resulted in a fair understanding of the overall strategy that these viruses employ to infect their hosts and to generate new virus particles. Papillomaviruses infect the basal layers of the epithelium, where the early viral genes are expressed and the viral DNA is replicated at a low level (1). As the infected cells migrate toward the skin surface and differentiate into keratinocytes, the viral DNA is replicated at high levels, viral capsid proteins are produced, and new virus particles are assembled (1). In contrast to other well-studied viruses, reproduction of the viral life cycle in vitro is difficult but can be achieved with low efficiency (2-4). Consequently, although the general functions of the virus-encoded polypeptides are known, many subtleties, including the consequences of modifications of the viral polypeptides, ranging from alternative splicing to posttranslational modifications, have been difficult to analyze and are poorly understood.The viral E1 and E2 proteins have been studied biochemically, genetically, and structurally and are among the best-studied polypeptides encoded by the papillomaviruses (5, 6). The E1 protein is a site-specific DNA binding protein that binds to the viral origin of DNA replication (ori) and opens the DNA duplex in preparation for initiation of DNA replication and also serves as the replicative DNA helicase (7-15). The E2 protein is a DNA binding transcription factor that can regulate viral transcription by binding to specific sites in the viral genome (16-21). The E2 protein is also required...

Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells

“…BPV E1 is therefore an in vitro substrate for purified CK2, and serines 94, 95, and 100 are likely target sites for this enzyme. This confirms similar data published recently [33]. …”

“…E1 is phosphorylated by several kinases, including Cdk2, CK2, MAPK, and PKC [4, 12, 15, 19–22, 25, 33, 40, 41]. Three BPV E1 phosphorylation sites (serines 94, 95, and 100) are within the bipartite nuclear localization signal (NLS), and all three are phosphorylated by CK2 [22, 33].…”

“…Three BPV E1 phosphorylation sites (serines 94, 95, and 100) are within the bipartite nuclear localization signal (NLS), and all three are phosphorylated by CK2 [22, 33]. CK2 is a ubiquitous serine/threonine kinase implicated in a wide range of essential cell functions including cell growth, apoptosis, gene expression and others.…”

“…These amino acids fall within the bipartite NLS of E1, and in the vicinity of other known regulatory phosphorylation sites [4, 20]. Algorithms that analyze protein phosphorylation predicted S94 and S95 as potential CK2 sites, which was recently confirmed [33]. If threonine 102 (T102, a Cdk2 phosphorylation site in E1) is phosphorylated, then S100 becomes a strongly predicted CK2 site [26].…”

Papillomavirus Replication

“…CK2 targets both E1 and E2 and decreases the DNA binding specificity of both proteins. These E1 and E2 PTMs keep replication activity low in viral maintenance, but the modification on E1 is lost via a caspase-dependent cleavage event during amplification (Schuck et al 2013). …”