Induction of the Bovine Papillomavirus Origin “Onion Skin”-Type DNA Replication at High E1 Protein Concentrations In Vivo

The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1^E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1^E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.Human papillomaviruses (HPVs) comprise a family of small DNA viruses that infect human keratinocytes in stratified epithelia at mucosal or cutaneous sites. Infections are typically subclinical or benign, but certain lesions can progress to cancers at a low frequency (81, 82). The viral genome consists of a double-stranded, circular DNA of approximately 7.9 kb, and it is normally maintained stably as autonomously replicating plasmids present at a low copy number in the basal cells of the epithelium. Vegetative reproduction depends on squamous differentiation, and elevated levels of viral transcription, DNA amplification, and virion morphogenesis occur sequentially in the lower and upper spinous strata of the epithelia. Progeny virus particles are then shed with the terminally differentiated, superficial cells. Because viral DNA replication depends largely on the cellular DNA machinery and entirely on host enzymes to generate deoxyribonucleoside triphosphate substrates, the viruses encode oncoproteins to reestablish an Sphase state in the postmitotic, differentiated cells (11,12,25).The HPV and bovine papillomavirus (BPV) replication origin recognition protein and transcription factor E2 bind as a dimer with high affinity and specificity to multiple copies of a consensus E2 binding site (E2BS), ACCGN 4 CGGT, in the viral origin of rep...

Section: Discussionmentioning

confidence: 99%

“…Another study showed that elevated levels of an HPV-1 E1 expression vector increased transient ori DNA replication, while increasing levels of an E2 expression vector reduced replication (76). A slight repression of BPV-1 DNA replication by elevated E2 expression was also reported (44).…”

mentioning

confidence: 97%

mRNA Splicing Regulates Human Papillomavirus Type 11 E1 Protein Production and DNA Replication

Deng

Jin

Lin

et al. 2003

“…It is possible that E1 unwinds chromosomal DNA, resulting in aberrant replicative intermediates and stalled replication forks and activation of both ATM and ATR pathways. High concentrations of the E1 protein have been shown to promote frequent loading of the helicase at the viral origin, resulting in "onion-skin" replication intermediates (35). In cells with integrated genomes, E1 expression induces amplification of integrated origins, leading to genomic instability and recruitment of the MRN complex, pATM, and pChk2 and phosphorylation of H2AX (25,26).…”

Section: Discussionmentioning

confidence: 99%

The Papillomavirus E1 Helicase Activates a Cellular DNA Damage Response in Viral Replication Foci

Sakakibara

Mitra

McBride

2011

170

234

The papillomavirus E1 and E2 proteins are essential for viral genome replication. E1 is a helicase that unwinds the viral origin and recruits host cellular factors to replicate the viral genome. E2 is a transcriptional regulator that helps recruit the E1 helicase to the origin and also plays a role in genome partitioning. We find that when coexpressed, the E1 and E2 proteins from several papillomavirus types localize to defined nuclear foci and result in growth suppression of the host cells. Growth suppression was due primarily to E1 protein function, and nuclear expression of E1 was accompanied by activation of a DNA damage response, resulting in phosphorylation of ATM, Chk2, and H2AX. Growth suppression and ATM activation required the ATPase and origin-specific binding functions of the E1 protein and resulted in active DNA repair, as evidenced by incorporation of nucleotide analogs and detection of free DNA ends. In the presence of the E2 protein, these activities became localized to nuclear foci. We postulate that these foci represent viral replication factories and that a cellular DNA damage response is activated to facilitate replication of viral DNA.Papillomaviruses (PVs) are double-stranded DNA viruses that infect cutaneous and mucosal epithelial cells of animals. Over 200 papillomavirus types have been reported to date, and phylogenetic classification indicates that there are at least 29 genera (1). Papillomavirus genomes consist of approximately 8 kb of double-stranded DNA, which typically contains seven to eight genes. Two of these genes encode replication proteins, E1 and E2. The E1 protein is a helicase that has been shown by structural and biochemical studies to be essential for initiation of viral DNA replication. The E1 protein by itself has low affinity for the viral origin of replication, which contains specific, palindromic E1 binding sites. However, the multifunctional E2 protein binds to specific sites adjacent to the E1 binding sites and helps recruit E1 in a cooperative manner. When loaded onto the viral replication origin, the E1 and E2 proteins recruit host replication factors such as RPA, topoisomerase I, and Pol␣/primase to initiate viral DNA replication (reviewed in reference 36). The E2 protein can function both as a transcriptional transactivator and repressor of viral early genes and for some papillomavirus types has also been shown to tether the viral genome to host chromatin to maintain and partition the extrachromosomal genomes.Eukaryotic cells have many different strategies to combat viral infection. Many viruses induce a cellular DNA damage response (DDR), either indirectly by virtue of viral DNA replicative intermediates that resemble damaged DNA or directly by viral protein function (reviewed in reference 51). The host cell induces the DNA damage response in an attempt to arrest cell growth and allow repair of genomic DNA damage, thus maintaining genomic stability. Two of the major regulators of the DNA damage response are the ATM (ataxia telangiectasia mutated) and the ATR (...

“…A model that would fit with the existing data is that E1 needs to be stockpiled for the vegetative part of the viral life cycle and that a high concentration of active E1 is deleterious for cell proliferation. It is well established that high-level expression of BPV E1 can cause problems for the host cell DNA (66). The inactivation of E1 by phosphorylation would allow stockpiling.…”

Section: Discussionmentioning

confidence: 99%

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

Schuck

Ruse

Stenlund

2013

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...