The papillomavirus E1 and E2 proteins are essential for viral genome

The papillomavirus E1 and E2 proteins are essential for viral genome replication. 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 51481-61-9 manufacture facilitate replication of viral DNA. INTRODUCTION 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 51481-61-9 manufacture 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 CDH1 to 51481-61-9 manufacture 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 (ATM and Rad3-related) kinases, which belong to the phosphoinositide-3-kinase-related protein kinases (PIKKs) (reviewed in reference 7). These kinases initiate a signal transduction cascade that activates many pathways and proteins to maintain genome integrity. While there is much overlap in the substrates of the ATM and ATR pathways, the ATR pathway is generally induced by single-stranded DNA at stalled replication forks whereas the ATM pathway is activated by double-strand DNA breaks (DSB) resulting from collapsed replication forks or ionizing irradiation (reviewed in reference 51). Among many of the substrates that are phosphorylated by activated ATM are H2AX and Chk2, while phosphorylation of Chk1 is associated with an induced ATR pathway. In the context of a viral infection, the host DNA damage response can be disadvantageous to viral production, and thus viruses have evolved mechanisms to overcome host cellular defenses and reprogram these responses for their own benefit (reviewed in references 4 and 51). For instance, in HIV infection, the Vpr accessory protein arrests cells in G2/M phases of the cell cycle and induces the ATR pathway by binding to chromatin and inducing Chk1 phosphorylation and the formation of phosphorylated H2AX (H2AX) and 53BP1 nuclear foci (33). This G2 arrest results in increased viral expression and production (17). In another example, detection of newly synthesized viral DNA during lytic replication of Epstein-Barr virus (EBV) results in an ATM DNA damage response (30). However, the ATM signaling cascade is modified by the viral BGLF4 kinase, resulting in promotion of an S-phase-like environment for viral DNA replication, inhibition of 51481-61-9 manufacture cellular DNA replication (29), and promotion of viral DNA circularization by homologous recombinational repair (31). Other small DNA viruses, such as polyomavirus and simian virus 40 (SV40), are similar to papillomaviruses and rely on cellular replication factors for DNA synthesis. SV40 and polyomaviruses reprogram and exploit the cellular DNA damage response for self-propagation (8, 19). SV40 large T antigen can induce DNA damage in the absence of the viral replication origin, thus activating the ATM, ATR, and Fanconi anemia (FA) homologous recombination pathways, which are required for efficient replication of SV40 (2). Recently, it was shown that high-risk oncogenic papillomaviruses also require the ATM pathway to efficiently amplify viral genomes in differentiated cells (39). However, the exact mechanism for the induction of DDR in cells harboring human papillomavirus (HPV) remains unclear. In this study we have shown that coexpression of the papillomavirus E1 and E2 proteins from various papillomavirus types resulted in cell growth suppression. The viral.

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