Identification du rôle et des modifications post-traductionnelles modulant l’export nucléaire de l’hélicase virale E1 au cours du cycle de réplication du virus du papillome humain

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin and the mucosa. HPV rely on two viral proteins, E1 and E2, to replicate and maintain their genome in the nucleus of infected cells. During replication, the E1 helicase is recruited to the origin of replication by E2 and is assembled into a double-hexamer that unwinds DNA ahead of the replication fork. E1 is comprised of a C-terminal enzymatic domain with ATPase/helicase activity, a central origin-binding domain and a N-terminal regulatory region that is required for viral DNA replication in vivo. The latter region of E1 contains a nuclear localization signal and a nuclear export signal that regulate its shuttling between the nucleus and cytoplasm. For bovine papillomavirus (BPV) E1, this shuttling was suggested to be controlled by the sumoylation of E1. In addition to the NES and NLS, the N-terminal region of E1 contains a conserved cyclin-binding motif that is required for the interaction of E1 with cyclin E/A-Cdk2. Cdk2 phosphorylation of E1 has been reported to control the nuclear export of E1 from BPV and HPV, albeit differently. In the first part of this study, we showed that although HPV E1 interacts with Ubc9, the conjugating enzyme of the sumoylation pathway, this pathway is not required for its accumulation in the nucleus. In the second part, we found that the nuclear accumulation of E1 is, instead, regulated by phosphorylation. Specifically, we found that Cdk2-dependent phosphorylation of conserved serines in the E1 N-terminal region inhibits the nuclear export of HPV E1. Furthermore, we reported that nuclear export is not essential to amplify the viral genome in differentiating keratinocytes but that it is required for its long-term maintenance in undifferentiated keratinocytes. Importantly, we found that the nuclear accumulation of E1 induces a S-phase arrest that is detrimental to cellular proliferation and that this anti-proliferative effect can be counteracted by the export of E1 from the nucleus to the cytoplasm. In the last part of this study, we showed that this arrest is dependent on the DNA- and ATP-binding activities of E1. Furthermore, we found that the cell cycle arrest induced by E1 is accompanied by the activation of a DNA damage response (DDR) dependent on the ATM (Ataxia Telangiectasia Mutated) pathway. However, these two events seem to be distinct since complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Importantly, we demonstrated that transient viral DNA replication still occurs in S-phase arrested cells, independently of the induction of a DDR and of the ATM kinase. Collectively, these data indicate that nuclear export of E1 is regulated by phosphorylation and not by sumoylation. They also revealed that nuclear export of E1 is essential for maintenance of the viral episome in keratinocytes, at least in part to limit its nuclear accumulation and prevent its detrimental effect on cellular proliferation and induction of a DDR

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes

Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance▿

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

Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA▿

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer

Proteasomal Degradation of the Papillomavirus E2 Protein Is Inhibited by Overexpression of Bromodomain-Containing Protein 4▿ ‡

The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation

Structure-Based Analysis of the Interaction between the Simian Virus 40 T-Antigen Origin Binding Domain and Single-Stranded DNA▿ †

The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT)12 is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication

CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

The cochaperone BCL2-associated athanogene 3 (BAG3), in complex with the heat shock protein HSPB8, facilitates mitotic rounding, spindle orientation, and proper abscission of daughter cells. BAG3 and HSPB8 mitotic functions implicate the sequestosome p62/SQSTM1, suggesting a role for protein quality control. However, the interplay between this chaperone-assisted pathway and the mitotic machinery is not known. Here, we show that BAG3 phosphorylation at the conserved T285 is regulated by CDK1 and activates its function in mitotic cell shape remodeling. BAG3 phosphorylation exhibited a high dynamic at mitotic entry and both a non-phosphorylatable BAG3T285A and a phosphomimetic BAG3T285D protein were unable to correct the mitotic defects in BAG3-depleted HeLa cells. We also demonstrate that BAG3 phosphorylation, HSPB8, and CDK1 activity modulate the molecular assembly of p62/SQSTM1 into mitotic bodies containing K63 polyubiquitinated chains. These findings suggest the existence of a mitotically regulated spatial quality control mechanism for the fidelity of cell shape remodeling in highly dividing cells