Cary A. Moody

Human papillomavirus oncoproteins: pathways to transformation.

An association between human papillomavirus (HPV) infection and the development of cervical cancer was initially reported over 30 years ago, and today there is overwhelming evidence that certain subtypes of HPV are the causative agents of these malignancies. The p53 and retinoblastoma proteins are well-characterized targets of the HPV E6 and E7 oncoproteins, but recent studies have shown that the alteration of additional pathways are equally important for transformation. These additional factors are crucial regulators of cell cycle progression, telomere maintenance, apoptosis and chromosomal stability. Understanding how HPV oncoproteins modify these activities provides novel insights into the basic mechanisms of oncogenesis.

Human papillomaviruses activate the ATM DNA damage pathway for viral genome amplification upon differentiation.

Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.

Human Papillomaviruses Recruit Cellular DNA Repair and Homologous Recombination Factors to Viral Replication Centers

ABSTRACT Human papillomaviruses (HPV) activate the ataxia telangiectasia mutated (ATM)-dependent DNA damage response to induce viral genome amplification upon epithelial differentiation. Our studies show that along with members of the ATM pathway, HPV proteins also localize factors involved in homologous DNA recombination to distinct nuclear foci that contain HPV genomes and cellular replication factors. These studies indicate that HPV activates the ATM pathway to recruit repair factors to viral genomes and allow for efficient replication.

Modulation of the Cell Growth Regulator mTOR by Epstein-Barr Virus-Encoded LMP2A

ABSTRACT Control of translation initiation is one means by which cells regulate growth and proliferation, with components of the protein-synthesizing machinery having oncogenic potential. Expression of latency protein LMP2A by the human tumor virus Epstein-Barr virus (EBV) activates phosphatidylinositol 3-kinase/Akt located upstream of an essential mediator of growth signals, mTOR (mammalian target of rapamycin). We show that mTOR is activated by expression of LMP2A in carcinoma cells, leading to wortmannin- and rapamycin-sensitive inhibition of the negative regulator of translation, eukaryotic initiation factor 4E-binding protein 1, and increased c-Myc protein translation. Intervention by this DNA tumor virus in cellular translational controls is likely to be an integral component of EBV tumorigenesis.

Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification

The life cycle of human papillomaviruses (HPVs) is linked to epithelial differentiation, with late viral events restricted to the uppermost stratified layers. Our studies indicated that HPV activates capases-3, -7, and -9 upon differentiation, whereas minimal activation was observed in differentiating normal keratinocytes. Activation occurred in the absence of significant levels of apoptosis, suggesting a potential role for caspases in the viral life cycle. In support of this, the addition of caspase inhibitors significantly impaired differentiation-dependent viral genome amplification. A conserved caspase cleavage motif was identified in the replication protein E1 (46DxxD49) that was targeted in vitro by both recombinant caspase-3 and caspase-7. Mutation of this site inhibited amplification of viral genomes, indicating that caspase cleavage is necessary for the productive viral life cycle. Our study demonstrates that HPV activates caspases upon differentiation to facilitate productive viral replication and represents a way by which HPV controls viral gene function in differentiating cells.

Nuclear Accumulation of the Papillomavirus E1 Helicase Blocks S-Phase Progression and Triggers an ATM-Dependent DNA Damage Response

ABSTRACT Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.

Nuclear export of human papillomavirus type 31 E1 is regulated by Cdk2 phosphorylation and required for viral genome maintenance.

ABSTRACT The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.

Human Papillomavirus E1 Helicase Interacts with the WD Repeat Protein p80 To Promote Maintenance of the Viral Genome in Keratinocytes

ABSTRACT Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1^E4 is necessary for association with CDK–cyclin complexes and G2/M cell cycle arrest of keratinocytes, but is not required for differentiation-dependent viral genome amplification or L1 capsid protein expression

The G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK-cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence ((43)RRLL(46)) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events.