Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling

NF-B is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-B with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IB␣ and subsequent activation of NF-B in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IB␣, which inhibits the activation of NF-B, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-B and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IB␣ mutant. Collectively, we propose that expression of E1 induces NF-B activation at least in part through the ATR-dependent DNA damage response and that NF-B in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-B may constitute a negative feedback loop. IMPORTANCE A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-B activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-B mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-B activity is involved in the establishment and maintenance of persistent HPV infection. Human papillomaviruses (HPVs) are a large family of nonenveloped, small DNA viruses with an approximately 8-kbp double-stranded circular genome that infect stratified squamous epithelium in various anatomical sites such as skin, the anogenital tract, and the oral cavity. Virus infection can cause hyperproliferative lesions, ranging from verrucae to cancer. To date, at least 180 types of HPVs have been cloned from clinical lesions and classified as either cutaneous or mucosal types based on their predilection for different sites of infection. A subset of mucosal HPVs, high-risk HPVs (HR-HPVs), such as HPV16, are strongly associated with anogenital cancers, including nearly 100% of cervical cancers and some proportion of head and neck cancers...

Section: Activation Of Nf-b By E1 Through Ddrsupporting

confidence: 93%

Activation of NF-κB by Human Papillomavirus 16 E1 Limits E1-Dependent Viral Replication through Degradation of E1

Nakahara

Tanaka

Ohno

et al. 2015

“…In the context of HPV, it is tempting to speculate that the presence of p80 at the replication fork may facilitate the recruitment of DDR proteins to promote viral DNA replication or, conversely, to alleviate problems linked with a stalled replication fork. However, we and others recently showed that DNA damage neither interferes with nor stimulates transient HPV DNA replication (18,31), suggesting that the effect of p80 in this context is independent of DDR signaling. Furthermore, Moody et al also observed, using the chemical inhibitor KU-55933, that the ATM pathway has no effect on the stable maintenance of the HPV episome in undifferentiated keratinocytes (40), in contrast to p80, whose interaction with E1 is essential for this process (12).…”

mentioning

confidence: 73%

Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

Lehoux

Fradet-Turcotte

Lussier-Price

et al. 2012

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1-and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. Human papillomaviruses (HPVs) are small double-stranded DNA viruses that replicate and cause hyperproliferative lesions within stratified epithelia (reviewed in references 26 and 34). Among the 100 described HPV genotypes, more than 30 infect the anogenital mucosa (3, 15). These mucosal types are classified as low-or high-risk viruses according to their capacity to cause benign or cancerous lesions, respectively. High-risk HPVs are the etiological agents of cervical cancer, the second leading cause of cancer in women worldwide (47). These viral types have also been linked with other malignancies of the genital tract and with the majority of anal cancers in both men and women (2,13,24,48). Specific high-risk types, and in particular HPV type 16 (HPV16), are also increasingly being associated with a subset of head and neck cancers (32).HPVs infect keratinocytes of the basal cell layer. Upon infection, the viral genome is established as a multicopy episome in the nuclei of these cells through several rounds of viral DNA replication. Maintenance of the viral episome at 50 to 100 copies in these undifferentiated cells is then achieved through low levels of DNA replication and is essential for the persistence of the infection, itself a risk factor for virus-induced carcinogenesis. As the infected cells differentiate and reach the upper layers of the ep...

“…In fact, it has been shown that papillomaviruses amplify their DNA in the G 2 phase of the cell cycle in cells that have already completed S phase (6) and that efficient amplification of the viral genome requires an activated ATM response in differentiated cells (39). The DNA damage response does not seem to interfere with HPV DNA replication, since a recent study showed that transient viral replication is not inhibited by DNA damage-inducing agents, indicating that E1 and E2 can replicate the viral genome in the presence of a DNA damage response (28).…”

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 (...