A small RNA system ensures accurate homologous pairing and unpaired silencing of meiotic chromosomes.

During meiosis, chromosomes with homologous partners undergo synaptonemal complex (SC)-mediated pairing, while the remaining unpaired chromosomes are heterochromatinized through unpaired silencing. Mechanisms underlying homolog recognition during SC formation are still unclear. Here, we show that the Caenorhabditis elegans Argonaute proteins, CSR-1 and its paralog CSR-2, interacting with 22G-RNAs, are required for synaptonemal complex formation with accurate homology. CSR-1 in nuclei and meiotic cohesin, constituting the SC lateral elements, were associated with nonsimple DNA repeats, including minisatellites and transposons, and weakly associated with coding genes. CSR-1-associated CeRep55 minisatellites were expressing 22G-RNAs and long noncoding (lnc) RNAs that colocalized with synaptonemal complexes on paired chromosomes and with cohesin regions of unpaired chromosomes. CeRep55 multilocus deletions reduced the efficiencies of homologous pairing and unpaired silencing, which were supported by the csr-1 activity. Moreover, CSR-1 and CSR-2 were required for proper heterochromatinization of unpaired chromosomes. These findings suggest that CSR-1 and CSR-2 play crucial roles in homology recognition, achieving accurate SC formation between chromosome pairs and condensing unpaired chromosomes by targeting repeat-derived lncRNAs.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

BACKGROUND: The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. METHODS: The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. RESULTS: It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. CONCLUSION: It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA.

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = - (23.5 - 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logß'pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logß'pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logß' unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.

Epigenetic control of rDNA loci in response to intracellular energy status.

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

SET-NUP214 and MLL cooperatively regulate the promoter activity of the HoxA10 gene.

SET-Nup214 is a recurrent fusion gene that is mainly observed in T-cell acute lymphoblastic leukemia (T-ALL). Dysregulation of homeobox (Hox) genes is frequently observed in patients with leukemia. Consistent with this, HoxA genes are upregulated in the SET-Nup214 + T-ALL cell line and patients. Although SET-Nup214 has been reported to be recruited to the promoter regions of HoxA genes, the detailed mechanisms of how SET-Nup214 specifically binds to HoxA gene promoters and regulates HoxA gene expression are not known. In this study, we demonstrated that SET-Nup214 interacts with MLL via the SET acidic region of SET-Nup214. SET-Nup214 and MLL cooperatively enhance the promoter activity of the HoxA10 gene. Neither the SET region alone nor the Nup214 region alone sufficiently enhanced the HoxA10 gene promoter. Our results indicated that the SET portion of the SET-Nup214-fusion protein is important for interactions with MLL and transcription enhancement of the HoxA10 gene. Thus, our study will contribute to the understanding of how SET-Nup214 and MLL disturb the expression of HoxA10 gene in leukemia.

Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication.

BACKGROUND: Replication of the influenza A viruses occurs in the cells through the viral RdRP consisting of PB1, PB2, and PA. Several cellular proteins are involved in these processes. This study aims to reveal the interaction between human SNX2 protein and the PA protein and the effects of the SNX2 on the virus replication. RESULTS: To identify potential host interacting proteins to the PA, yeast two-hybrid assay was carried out with HEK293 cell cDNA library and the PA as a bait. We focused on SNX2 protein, which interacts with the PA in the yeast cells. By using the co-immunoprecipitation assays, it has been demonstrated that the amino-terminal part of the PA was important for binding to the SNX2. Immunolocalization of the proteins in HeLa cells supported this interaction. Knockdown of the SNX2 with siRNA in the cells resulted in a significant increase in both viral transcripts and virus growth. However, the increase of SNX2 in transfected cells didn't cause a significant change in the viral RdRP activity in minireplicon assay. This may suggest that the negative effect of SNX2 on the virus replication could be saturated with its authentic intra-cellular amount. CONCLUSIONS: This study revealed that the SNX2 and PA protein interact with each other in both yeast and HEK293 cells, and the SNX2 has a negative regulatory function on the virus replication. However, more knowledge is required to elucidate the action mechanism of the SNX2 on the influenza A virus replication at the molecular level.

Long noncoding RNA NEAT1-dependent SFPQ relocation from promoter region to paraspeckle mediates IL8 expression upon immune stimuli.

Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.

Influenza A Virus Infection Triggers Pyroptosis and Apoptosis of Respiratory Epithelial Cells through the Type I Interferon Signaling Pathway in a Mutually Exclusive Manner.

Respiratory epithelial cell death by influenza virus infection is responsible for the induction of inflammatory responses, but the exact cell death mechanism is not understood. Here we showed that influenza virus infection induces apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced only in malignant tumor cells infected with influenza virus. In human precancerous respiratory epithelial cells (PL16T), the number of apoptotic cells increased at early phases of infection, but pyroptotic cells were observed at late phases of infection. These findings suggest that apoptosis is induced at early phases of infection but the cell death pathway is shifted to pyroptosis at late phases of infection. We also found that the type I interferon (IFN)-mediated JAK-STAT signaling pathway promotes the switch from apoptosis to pyroptosis by inhibiting apoptosis possibly through the induced expression of the Bcl-xL anti-apoptotic gene. Further, the inhibition of JAK-STAT signaling repressed pyroptosis but enhanced apoptosis in infected PL16T cells. Collectively, we propose that type I IFN signaling pathway triggers pyroptosis but not apoptosis in the respiratory epithelial cells in a mutually exclusive manner to initiate proinflammatory responses against influenza virus infection.IMPORTANCE Respiratory epithelium functions as a sensor of infectious agents to initiate inflammatory responses along with cell death. However, the exact cell death mechanism responsible for inflammatory responses by influenza virus infection is still unclear. We showed that influenza virus infection induced apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced at early phases of infection, but the cell death pathway was shifted to pyroptosis at late phases of infection under the regulation of type I IFN signaling to promote proinflammatory cytokine production. Taken together, our results indicate that the type I IFN signaling pathway plays an important role to induce pyroptosis but represses apoptosis in the respiratory epithelial cells to initiate proinflammatory responses against influenza virus infection.

Efficient DNA binding of NF-κB requires the chaperone-like function of NPM1.

NPM1/nucleophosmin is frequently overexpressed in various tumors, although the oncogenic role of NPM1 remains unclear. Here we revealed the link between NPM1 and nuclear factor-κB (NF-κB), a master regulator of inflammation. We found that NPM1 knockdown decreased NF-κB-mediated transcription of selected target genes by decreasing the recruitment of NF-κB p65 to the gene promoters. NPM1 is directly associated with the DNA binding domain of p65 to enhance its DNA binding activity without being a part of the DNA-NF-κB complex. This result suggests that NF-κB requires the chaperone-like function of NPM1 for DNA binding. Furthermore, we demonstrated that NPM1 was required for efficient inflammatory gene expression induced by tumor necrosis factor alpha (TNF-α) and lipopolysaccharide in fibroblasts and macrophages. The NF-κB-mediated invasion of breast cancer cells was significantly decreased by NPM1 knockdown. Our study suggests a novel mechanistic insight into the NF-κB-mediated transcription and an oncogenic role of NPM1 in both tumor cells and the tumor micro-environment through the regulation of NF-κB.

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments.

In adenoviral virions, the genome is organized into a chromatin-like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication-related factors. We show de novo formation of a subnuclear domain, which we termed Virus-induced Post-Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.

Current state of therapeutic development for rare cancers in Japan, and proposals for improvement.

This article discusses current obstacles to the rapid development of safe and effective treatments for rare cancers, and considers measures required to overcome these challenges. In order to develop novel clinical options for rare cancers, which tend to remain left out of novel therapeutic development because of their paucity, efficient recruitment of eligible patients, who tend to be widely dispersed across the country and treated at different centers, is necessary. For this purpose, it is important to establish rare cancer registries that are linked with clinical studies, to organize a central pathological diagnosis system and biobanks for rare cancers, and to consolidate patients with rare cancers to facilities that can conduct clinical studies meeting international standards. Establishing an all-Japan cooperative network is essential. Clinical studies of rare cancers have considerable limitations in study design and sample size as a result of paucity of eligible patients and, as a result, the level of confirmation of the efficacy and safety shown by the studies is relatively low. Therefore, measures to alleviate these weaknesses inherent to external conditions need to be explored. It is also important to reform the current research environment in order to develop world-leading treatment for rare cancers, including promotion of basic research, collaboration between industry and academia, and improvement of the infrastructure for clinical studies. Collaboration among a wide range of stakeholders is required to promote the clinical development of treatment for rare cancers under a nationwide consensus.

Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease.

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA-binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.

Histone H1 chaperone activity of TAF-I is regulated by its subtype-dependent intramolecular interaction.

Linker histone H1 is involved in the regulation of gene activity through the maintenance of higher-order chromatin structure. Previously, we have shown that template activating factor-I (TAF-I or protein SET) is involved in linker histone H1 dynamics as a histone H1 chaperone. In human and murine cells, two TAF-I subtypes exist, namely TAF-Iα and TAF-Iß. TAF-I has a highly acidic amino acid cluster in its C-terminal region and forms homo- or heterodimers through its dimerization domain. Both dimer formation and the C-terminal region of TAF-I are essential for the histone chaperone activity. TAF-Iα exhibits less histone chaperone activity compared with TAF-Iß even though TAF-Iα and ß differ only in their N-terminal regions. However, it is unclear how subtype-specific TAF-I activities are regulated. Here, we have shown that the N-terminal region of TAF-Iα autoinhibits its histone chaperone activity via intramolecular interaction with its C-terminal region. When the interaction between the N- and C-terminal regions of TAF-Iα is disrupted, TAF-Iα shows a histone chaperone activity similar to that of TAF-Iß. Taken together, these results provide mechanistic insights into the concept that fine tuning of TAF-I histone H1 chaperone activity relies on the subtype compositions of the TAF-I dimer.

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain.

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128.

Analysis by metadynamics simulation of binding pathway of influenza virus M2 channel blockers.

M2 protein of influenza A virus is a proton channel spanning the viral envelope. Activity of this proton channel is required for uncoating of viral particles and equilibrating the pH across the trans Golgi apparatus, which prevents conformational change in hemagglutinin. Amantadine, an anti-influenza A virus drug, inhibits M2 proton channel activity by binding to the channel pore; however, most currently circulating influenza A viruses are amantadine-resistant. The most prevalent resistant mutation is a substitution from Ser31 to Asn31 in M2. Further atomistic analysis of ligand-M2 complexes is needed to provide new approaches for the design of novel M2 channel blockers. Here, the free energy profiles of the binding kinetics of M2 channel blockers were examined by well-tempered metadynamics simulations and it was found that amantadine first binds to Asp24 of S31 M2 and forms a metastable conformation. In contrast, the free energy profiles of adamantyl bromothiophene dual inhibitor with either S31 M2 or N31 M2 are broad funnel-shaped curves, suggesting that adamantyl bromothiophene does not form metastable complexes with M2. The trajectory of well-tempered metadynamics simulations revealed that steric hindrance between adamantyl bromothiophene and S31 M2 interrupts formation of a metastable conformation at Asp24 and that a halogen bond between the bromine atom and N31 is responsible for pulling down the ligand to the channel pore of N31 M2 in the absence of a metastable state. Binding pathways of M2 channel blockers to M2 are here proposed on the basis of these findings; they may provide new approaches to designing further M2 channel blockers.

The messenger RNA decapping and recapping pathway in Trypanosoma.

The 5' terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5'-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5'-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA ß-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5'-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.

Function of Nup98 subtypes and their fusion proteins, Nup98-TopIIß and Nup98-SETBP1 in nuclear-cytoplasmic transport.

Nup98 is a component of the nuclear pore complex. The nup98-fusion genes derived by chromosome translocations are involved in hematopoietic malignancies. Here, we investigated the functions of Nup98 isoforms and two unexamined Nup98-fusion proteins, Nup98-TopIIß and Nup98-SETBP1. We first demonstrated that two Nup98 isoforms are expressed in various mouse tissues and similarly localized in the nucleus and the nuclear envelope. We also showed that Nup98-TopIIß and Nup98-SETBP1 are localized in the nucleus and partially co-localized with full-length Nup98 and a nuclear export receptor XPO1. We demonstrated that Nup98-TopIIß and Nup98-SETBP1 negatively regulate the XPO1-mediated protein export. Our results will contribute to the understanding of the molecular mechanism by which the Nup98-fusion proteins induce tumorigenesis.

C-terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A-H2B, but not H3-H4.

Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)2 tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

UNLABELLED: Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. IMPORTANCE: The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes.