BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

The BRCA1 BRCT promotes antisense RNA production and double-stranded RNA formation to suppress ribosomal R-loops.

R-loops, or RNA:DNA hybrids, can induce DNA damage, which requires DNA repair factors including breast cancer type 1 susceptibility protein (BRCA1) to restore genomic integrity. To date, several pathogenic mutations have been found within the tandem BRCA1 carboxyl-terminal (BRCT) domains that mediate BRCA1 interactions with proteins and DNA in response to DNA damage. Here, we describe a nonrepair role of BRCA1 BRCT in suppressing ribosomal R-loops via two mechanisms. Through its RNA binding and annealing activities, BRCA1 BRCT facilitates the formation of double-stranded RNA between ribosomal RNA (rRNA) and antisense-rRNA (as-rRNA), hereby minimizing rRNA hybridization to ribosomal DNA to form R-loops. BRCA1 BRCT also promotes RNA polymerase I-dependent transcription of as-rRNA to enhance double-stranded rRNA (ds-rRNA) formation. In addition, BRCA1 BRCT-mediated as-rRNA production restricts rRNA maturation in unperturbed cells. Hence, impairing as-rRNA transcription and ds-rRNA formation due to BRCA1 BRCT deficiency deregulates rRNA processing and increases ribosomal R-loops and DNA breaks. Our results link ribosomal biogenesis dysfunction to BRCA1-associated genomic instability.

TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks.

In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription-replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.

The DUF328 family member YaaA is a DNA-binding protein with a novel fold.

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nm YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation.

UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

BGLF4 kinase modulates the structure and transport preference of the nuclear pore complex to facilitate nuclear import of Epstein-Barr virus lytic proteins.

UNLABELLED: BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin ß was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses. IMPORTANCE: During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.

The ESCRT machinery is recruited by the viral BFRF1 protein to the nucleus-associated membrane for the maturation of Epstein-Barr Virus.

The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.

Epstein-Barr virus protein kinase BGLF4 targets the nucleus through interaction with nucleoporins.

BGLF4 of Epstein-Barr virus (EBV) encodes a serine/threonine protein kinase that phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of viral nucleocapsids. BGLF4 is expressed predominantly in the nucleus at early and late stages of virus replication, while a small portion of BGLF4 is distributed in the cytoplasm at the late stage of virus replication and packaged into the virion. Here, we analyzed systematically the functional domains crucial for nuclear localization of BGLF4 and found that both the N and C termini play important modulating roles. Analysis of amino acid substitution mutants revealed that the C terminus of BGLF4 does not contain a conventional nuclear localization signal (NLS). Additionally, deletion of the C-terminal putative helical regions at amino acids 386 to 393 and 410 to 419 diminished the nuclear translocation of BGLF4, indicating that the secondary structure of the C terminus is important for the localization of BGLF4. The green fluorescent protein-fused wild-type or C-terminal helical regions of BGLF4 associate with phenylalanine/glycine repeat-containing nucleoporins (Nups) in nuclear envelope fractionation. Both coimmunoprecipitation and in vitro pull-down assays further demonstrated that BGLF4 binds to Nup62 and Nup153. Remarkably, nuclear import assay with permeabilized HeLa cells demonstrated that BGLF4 translocated into nucleus independent of cytosolic factors. Data presented here suggest that BGLF4 employs a novel mechanism through direct interactions with nucleoporins for its nuclear targeting.

Epstein-Barr virus BGLF4 kinase downregulates NF-κB transactivation through phosphorylation of coactivator UXT.

Epstein-Barr virus (EBV) BGLF4 is a member of the conserved herpesvirus kinases that regulate multiple cellular and viral substrates and play an important role in the viral lytic cycles. BGLF4 has been found to phosphorylate several cellular and viral transcription factors, modulate their activities, and regulate downstream events. In this study, we identify an NF-κB coactivator, UXT, as a substrate of BGLF4. BGLF4 downregulates not only NF-κB transactivation in reporter assays in response to tumor necrosis factor alpha (TNF-α) and poly(I·C) stimulation, but also NF-κB-regulated cellular gene expression. Furthermore, BGLF4 attenuates NF-κB-mediated repression of the EBV lytic transactivators, Zta and Rta. In EBV-positive NA cells, knockdown of BGLF4 during lytic progression elevates NF-κB activity and downregulates the activity of the EBV oriLyt BHLF1 promoter, which is the first promoter activated upon lytic switch. We show that BGLF4 phosphorylates UXT at the Thr3 residue. This modification interferes with the interaction between UXT and NF-κB. The data also indicate that BGLF4 reduces the interaction between UXT and NF-κB and attenuates NF-κB enhanceosome activity. Upon infection with short hairpin RNA (shRNA) lentivirus to knock down UXT, a spontaneous lytic cycle was observed in NA cells, suggesting UXT is required for maintenance of EBV latency. Overexpression of wild-type, but not phosphorylation-deficient, UXT enhances the expression of lytic proteins both in control and UXT knockdown cells. Taking the data together, transcription involving UXT may also be important for EBV lytic protein expression, whereas BGLF4-mediated phosphorylation of UXT at Thr3 plays a critical role in promoting the lytic cycle.

DNA replication initiation factor RECQ4 possesses a role in antagonizing DNA replication initiation.

Deletion of the conserved C-terminus of the Rothmund-Thomson syndrome helicase RECQ4 is highly tumorigenic. However, while the RECQ4 N-terminus is known to facilitate DNA replication initiation, the function of its C-terminus remains unclear. Using an unbiased proteomic approach, we identify an interaction between the RECQ4 N-terminus and the anaphase-promoting complex/cyclosome (APC/C) on human chromatin. We further show that this interaction stabilizes APC/C co-activator CDH1 and enhances APC/C-dependent degradation of the replication inhibitor Geminin, allowing replication factors to accumulate on chromatin. In contrast, the function is blocked by the RECQ4 C-terminus, which binds to protein inhibitors of APC/C. A cancer-prone, C-terminal-deleted RECQ4 mutation increases origin firing frequency, accelerates G1/S transition, and supports abnormally high DNA content. Our study reveals a role of the human RECQ4 C-terminus in antagonizing its N-terminus, thereby suppressing replication initiation, and this suppression is impaired by oncogenic mutations.

Molecular Mechanisms of the RECQ4 Pathogenic Mutations.

The human RECQ4 gene encodes an ATP-dependent DNA helicase that contains a conserved superfamily II helicase domain located at the center of the polypeptide. RECQ4 is one of the five RECQ homologs in human cells, and its helicase domain is flanked by the unique amino and carboxyl termini with sequences distinct from other members of the RECQ helicases. Since the identification of the RECQ4 gene in 1998, multiple RECQ4 mutations have been linked to the pathogenesis of three clinical diseases, which are Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO. Patients with these diseases show various developmental abnormalities. In addition, a subset of RECQ4 mutations are associated with high cancer risks, especially for osteosarcoma and/or lymphoma at early ages. The discovery of clinically relevant RECQ4 mutations leads to intriguing questions: how is the RECQ4 helicase responsible for preventing multiple clinical syndromes? What are the mechanisms by which the RECQ4 disease mutations cause tissue abnormalities and drive cancer formation? Furthermore, RECQ4 is highly overexpressed in many cancer types, raising the question whether RECQ4 acts not only as a tumor suppressor but also an oncogene that can be a potential new therapeutic target. Defining the molecular dysfunctions of different RECQ4 disease mutations is imperative to improving our understanding of the complexity of RECQ4 clinical phenotypes and the dynamic roles of RECQ4 in cancer development and prevention. We will review recent progress in examining the molecular and biochemical properties of the different domains of the RECQ4 protein. We will shed light on how the dynamic roles of RECQ4 in human cells may contribute to the complexity of RECQ4 clinical phenotypes.

Pathogenic mutations reveal a role of RECQ4 in mitochondrial RNA:DNA hybrid formation and resolution.

The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein-protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.

SUMO2 conjugation of PCNA facilitates chromatin remodeling to resolve transcription-replication conflicts.

During DNA synthesis, DNA replication and transcription machinery can collide, and the replication fork may temporarily dislodge RNA polymerase II (RNAPII) to resolve the transcription-replication conflict (TRC), a major source of endogenous DNA double-strand breaks (DSBs) and common fragile site (CFS) instability. However, the mechanism of TRC resolution remains unclear. Here, we show that conjugation of SUMO2, but not SUMO1 or SUMO3, to the essential replication factor PCNA is induced on transcribed chromatin by the RNAPII-bound helicase RECQ5. Proteomic analysis reveals that SUMO2-PCNA enriches histone chaperones CAF1 and FACT in the replication complex via interactions with their SUMO-interacting motifs. SUMO2-PCNA enhances CAF1-dependent histone deposition, which correlates with increased histone H3.1 at CFSs and repressive histone marks in the chromatin to reduce chromatin accessibility. Hence, SUMO2-PCNA dislodges RNAPII at CFSs, and overexpressing either SUMO2-PCNA or CAF1 reduces the incidence of DSBs in TRC-prone RECQ5-deficient cells.

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment.

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Glycogen synthase kinase 3 negatively regulates IFN regulatory factor 3 transactivation through phosphorylation at its linker region.

Upon virus infection, the host innate immune response is initiated through the activation of IFN regulatory factor 3 (IRF3) and NF-κB signaling pathways to induce IFN production. Previously, we demonstrated EBV BGLF4 kinase suppresses IRF3 function in a kinase activity-dependent manner. The replacement of Ser123, Ser173 and Thr180 into alanines at the proline-rich linker region of IRF3 abolishes BGLF4-mediated suppression. In this study, we show that BGLF4 phosphorylates glutathione-S-transferase (GST)-IRF3(110-202), but not GST-IRF3(110-202)3A mutant (S123/S173/T180A) in vitro. Compared with activation mimicking mutant IRF3(5D), the phosphorylation-defective IRF3(5D)3A shows a higher transactivation activity in reporter assays, whereas the phosphorylation-mimicking IRF3(5D)2D1E, with Ser123 and Ser173 mutated to aspartate and Thr180 to glutamate, has a much lower activity. To explore whether similar cellular regulation also exists in the absence of virus infection, candidate cellular kinases were predicted and the transactivation activity of IRF3 was examined with various kinase inhibitors. Glycogen synthase kinase 3 (GSK3) inhibitor LiCl specifically enhanced both IRF3(5D) and wild type IRF3 activity, even without stimulation. Expression of constitutive active GSK3ß(S9A) represses LiCl-mediated enhancement of IRF3 transactivation activity. In vitro, both GSK3α and GSK3ß phosphorylate IRF3 at the linker region. Collectively, data here suggest GSK3 phosphorylates IRF3 linker region in a way similar to viral kinase BGLF4.