Auto-Regulatory mechanism of enzyme activity by the nuclear localization signal of lysine specific demethylase 1.

The N-terminal region of the human Lysine Specific Demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple post-translational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N-terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N-terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1's own electropositive NLS amino acids 107-120 inhibit demethylation activity on a model Histone 3 lysine 4 di-methyl (H3K4me2) peptide (Kiapp ∼ 3.3 µM) and H3K4me2 nucleosome substrates (IC50 ∼ 30.4 µM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, IDRs, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.

Nanoassemblies designed for efficient nuclear targeting.

One of the key aspects of coping efficiently with complex pathological conditions is delivering the desired therapeutic compounds with precision in both space and time. Therefore, the focus on nuclear-targeted delivery systems has emerged as a promising strategy with high potential, particularly in gene therapy and cancer treatment. Here, we explore the design of supramolecular nanoassemblies as vehicles to deliver specific compounds to the nucleus, with the special focus on polymer and peptide-based carriers that expose nuclear localization signals. Such nanoassemblies aim at maximizing the concentration of genetic and therapeutic agents within the nucleus, thereby optimizing treatment outcomes while minimizing off-target effects. A complex scenario of conditions, including cellular uptake, endosomal escape, and nuclear translocation, requires fine tuning of the nanocarriers' properties. First, we introduce the principles of nuclear import and the role of nuclear pore complexes that reveal strategies for targeting nanosystems to the nucleus. Then, we provide an overview of cargoes that rely on nuclear localization for optimal activity as their integrity and accumulation are crucial parameters to consider when designing a suitable delivery system. Considering that they are in their early stages of research, we present various cargo-loaded peptide- and polymer nanoassemblies that promote nuclear targeting, emphasizing their potential to enhance therapeutic response. Finally, we briefly discuss further advancements for more precise and effective nuclear delivery.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

Nuclear localization signal in nuclear receptor VDR facilitates the mitotic genome bookmarking by involving distinct amino acid residues.

Mitotic genome-bookmarking preserves epigenetic information, re-establishing progenitor's gene expression profile through transcription factors, chromatin remodelers, and histone modifiers, thereby regulating cell fate and lineage commitment post-mitotically in progeny cells. Our recent study revealed that the constitutive association of VDR with mitotic chromatin involves its DNA-binding domain. However, amino acid residues in this domain, crucial for genome bookmarking, remain elusive. This study demonstrates that nuclear localization signal (NLS) residues between 49 and 55 amino acids in VDR are essential for receptor-chromatin interaction during mitosis. Furthermore, it is revealed that both bipartite nature of VDR-NLS region and N-terminally located positively charged arginine residues are critical for its 'genome-bookmarking' property. Since mitotic chromatin association of heterodimeric partner RXR depends on VDR-chromatin association, interventions in VDR binding also abort RXR-chromatin interaction. Overall, this study documents the mechanistic details underlying VDR-chromatin interactions in genome-bookmarking behavior, potentially aiding in comprehending VDR-mediated diseases attributed to certain SNPs.

Multiple functions of the nonstructural protein 3D in picornavirus infection.

3D polymerase, also known as RNA-dependent RNA polymerase, is encoded by all known picornaviruses, and their structures are highly conserved. In the process of picornavirus replication, 3D polymerase facilitates the assembly of replication complexes and directly catalyzes the synthesis of viral RNA. The nuclear localization signal carried by picornavirus 3D polymerase, combined with its ability to interact with other viral proteins, viral RNA and cellular proteins, indicate that its noncatalytic role is equally important in viral infections. Recent studies have shown that 3D polymerase has multiple effects on host cell biological functions, including inducing cell cycle arrest, regulating host cell translation, inducing autophagy, evading immune responses, and triggering inflammasome formation. Thus, 3D polymerase would be a very valuable target for the development of antiviral therapies. This review summarizes current studies on the structure of 3D polymerase and its regulation of host cell responses, thereby improving the understanding of picornavirus-mediated pathogenesis caused by 3D polymerase.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.

Effect of Different Karyophilic Peptides on Physical Characteristics and In Vitro Transfection Efficiency of Chitosan-Plasmid Nanoparticles as Nonviral Gene Delivery Systems.

A strategy to increase the transfection efficiency of chitosan-based nanoparticles for gene therapy is by adding nuclear localization signals through karyophilic peptides. Here, the effect of the length and sequence of these peptides and their interaction with different plasmids on the physical characteristics and biological functionality of nanoparticles is reported. The karyophilic peptides (P1 or P2) were used to assemble nanoparticles by complex coacervation with pEGFP-N1, pQBI25 or pSelect-Zeo-HSV1-tk plasmids, and chitosan. Size, polydispersity index, zeta potential, and morphology, as well as in vitro nucleus internalization and transfection capability of nanoparticles were determined. The P2 nanoparticles resulted smaller compared to the ones without peptides or P1 for the three plasmids. In general, the addition of either P1 or P2 did not have a significant impact on the polydispersity index and the zeta potential. P1 and P2 nanoparticles were localized in the nucleus after 30 min of exposure to HeLa cells. Nevertheless, the presence of P2 in pEGFP-N1 and pQBI25 nanoparticles raised their capability to transfect and express the green fluorescent protein. Thus, karyophilic peptides are an efficient tool for the optimization of nonviral vectors for gene delivery; however, the sequence and length of peptides have an impact on characteristics and functionality of nanoparticles.

DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection.

Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Intracellular delivery of nuclear localization sequence peptide mitigates COVID-19 by inhibiting nuclear transport of inflammation-associated transcription factors.

The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), can trigger dysregulated immune responses known as the cytokine release syndrome (CRS), leading to severe organ dysfunction and respiratory distress. Our study focuses on developing an improved cell-permeable nuclear import inhibitor (iCP-NI), capable of blocking the nuclear transport of inflammation-associated transcription factors, specifically nuclear factor kappa B (NF-κB). By fusing advanced macromolecule transduction domains and nuclear localization sequences from human NF-κB, iCP-NI selectively interacts with importin α5, effectively reducing the expression of proinflammatory cytokines. In mouse models mimic SARS-CoV-2-induced pneumonitis, iCP-NI treatment demonstrated a significant decrease in mortality rates by suppressing proinflammatory cytokine production and immune cell infiltration in the lungs. Similarly, in hamsters infected with SARS-CoV-2, iCP-NI effectively protected the lung from inflammatory damage by reducing tumor necrosis factor-α, interleukin-6 (IL-6), and IL-17 levels. These promising results highlight the potential of iCP-NI as a therapeutic approach for COVID-19-related lung complications and other inflammatory lung diseases.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha.

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Functional Divergence and Origin of the Vertebrate Praja Family.

The Praja family is an E3 ubiquitin ligase, promoting polyubiquitination and subsequent degradation of substrates. It comprises two paralogs, praja1 and praja2. Prior research suggests these paralogs have undergone functional divergence, with examples, such as their distinct roles in neurite outgrowth. However, the specific evolutionary trajectories of each paralog remain largely unexplored preventing mechanistic understanding of functional differences between paralogs. Here, we investigated the phylogeny and divergence of the vertebrate Praja family through molecular evolutionary analysis. Phylogenetic examination of the vertebrate praja revealed that praja1 and praja2 originated from the common ancestor of placentals via gene duplication, with praja1 evolving at twice the rate of praja2 shortly after the duplication. Moreover, a unique evolutionary trajectory for praja1 relative to other vertebrate Praja was indicated, as evidenced by principal component analysis on GC content, codon usage frequency, and amino acid composition. Subsequent motif/domain comparison revealed conserved N terminus and C terminus in praja1 and praja2, together with praja1-specific motifs, including nuclear localization signal and Ala-Gly-Ser repeats. The nuclear localization signal was demonstrated to be functional in human neuroblastoma SH-SY5Y cells using deletion mutant, while praja2 was exclusively expressed in the nucleus. These discoveries contribute to a more comprehensive understanding of the Praja family's phylogeny and suggest a functional divergence between praja1 and praja2. Specifically, the shift of praja1 into the nucleus implies the degradation of novel substrates located in the nucleus as an evolutionary consequence.

Insights on the nuclear shuttling of H2A-H2B histone chaperones.

All cellular processes that involve the unwinding of DNA also lead to the systematic shuttling of histones. Histone shuttling across the nuclear membrane is facilitated by a class of proteins known as - histone chaperones. Histone chaperones are classified based on their binding to H3/H4 histones or H2A/H2B histones. During the shuttling process, two types of signals - NLS and NES are recognized by the nuclear transport proteins. However, this is the nuclear transport protein and the mechanism of signal recognition by the protein is still unknown. Thus, in this piece of work, the NLS and NES signals are predicted on important H2A/H2B binding histone chaperones. In addition, cellular localization and potential DNA binding regions of histone chaperones are predicted. Mapping of predicted regions on the histone chaperone's structure suggested that the critical binding regions mainly lie on the disordered region of the histone chaperones. NLS and NES are present in the N- and C-terminal of the histone chaperones. Most histone chaperones contain bipartiate NLS signals. This article sheds light on the crucial aspect that in addition of being directly engaged in nucleosome synthesis and disassembly in vivo, histone chaperone also performs various specific roles via histone binding activity.

Importance of nuclear localization signal-fused Cas9 in the production of genome-edited mice via embryo electroporation.

Previously, to generate genome-edited animals by introducing CRISPR-associated protein 9 (Cas9) into embryos, we developed the Technique for Animal Knockout system by Electroporation (TAKE). Additionally, by fluorescently labeling Cas9, we successfully visualized the Cas9 introduced into the pronuclei of embryos; however, whether Cas9 was introduced directly into the pronuclei by electric pulse or transferred from the cytoplasm by nuclear localization signal (NLS) remained unknown. Herein, we evaluated the localization of Cas9 with (Cas9-NLS) or without NLS (Cas9-noNLS) in mice embryos following electroporation by fusing them with GFP. Furthermore, we visually studied their effects on genome-editing rates in offspring by targeting tyrosinase gene. Fluorescence intensity in pronuclei of Cas9-NLS-electroporated embryos and genome-editing rates of offspring were significantly higher than those of Cas9-noNLS-electroporated embryos. Furthermore, fluorescence in Cas9-NLS-electroporated embryos in which pronuclei had not yet appeared 2.5 h after insemination was observed in the pronuclei of embryos appearing 3.5 h after electroporation. We demonstrated the effective transportation of Cas9 from the cytoplasm to pronuclei by the NLS following TAKE, which resulted in increased genome-editing rates in offspring. The TAKE along with fluorescently labeled nucleases can be used to verify nuclease delivery into individual embryos prior to embryo transfer for efficiently producing genome-edited animals.

Suppression of SAMSN1 contributes to neuroprotection in neonatal rats suffering from hypoxic-ischemic encephalopathy injury.

This article aims to detect the effect of SAM domain, SH3 domain, and nuclear localization signal 1 (SAMSN1) in neonatal rats with neurological dysfunction induced by hypoxia and ischemia (HI). The HI model was created using 7-day postnatal rats. Zea-longa score was utilized to validate the neurological injury after HI. Then, the differentially expressed genes (DEGs) were detected by gene sequencing and bioinformatics analysis methods. The oxygen and glucose deprivation (OGD) models were established in the SY5Y cells and fetal human cortical neurons. In addition, SAMSN1-small interfering RNA, methyl thiazolyl tetrazolium assay, and cell growth curve were employed to evaluate the cell viability variation. Obviously, Zea-longa scores increased in rats with HI insult. Subsequently, SAMSN1 was screened out, and it was found that SAMSN1 was strikingly upregulated in SY5Y cells and fetal neurons post-OGD. Interestingly, we found that SAMSN1 silencing could markedly enhance cell viability and cell growth after OGD. These data suggested that downregulation of SAMSN1 may exert a neuroprotective effect on damaged neurons after HI by improving cell viability and cell survival, which provides a potential theoretical basis for clinical trials in the future to treat neonatal hypoxic-ischemic encephalopathy.

Improved biocompatibility of dendrimer-based gene delivery by histidine-modified nuclear localization signals.

Polyamidoamine (PAMAM) dendrimers have been explored as an alternative to polyethylenimine (PEI) as a gene delivery carrier because of their relatively low cytotoxicity and excellent biocompatibility. The transfection efficiency of PAMAM dendrimers can be improved by the addition of nuclear localization signal (NLS), a positively charged peptide sequence recognized by cargo proteins in the cytoplasm for nuclear transport. However, increased positive charges from NLS can cause damage to the cytoplasmic and mitochondrial membranes and lead to reactive oxygen species (ROS)-induced cytotoxicity. This negative effect of NLS can be negated without a significant reduction in transfection efficiency by adding histidine, an essential amino acid known as a natural antioxidant, to NLS. However, little is known about the exact mechanism by which histidine reduces cytotoxicity of NLS-modified dendrimers. In this study, we selected cystamine core PAMAM dendrimer generation 2 (cPG2) and conjugated it with NLS derived from Merkel cell polyomavirus large T antigen and histidine (n = 0-3) to improve transfection efficiency and reduce cytoxicity. NLS-modified cPG2 derivatives showed similar or higher transfection efficiency than PEI 25 kDa in NIH3T3 and human mesenchymal stem cells (hMSC). The cytotoxicity of NLS-modified cPG2 derivatives was substantially lower than PEI 25 kDa and was further reduced as the number of histidine in NLS increased. To understand the mechanism of cytoprotective effect of histidine-conjugated NLS, we examined ROS scavenging, hydroxyl radical generation and mitochondrial membrane potential as a function of the number of histidine in NLS. As the number of hisidine increased, cPG2 scavenged ROS more effectively as evidenced by the hydroxyl radical antioxidant capacity (HORAC) assay. This was consistent with the reduced intracellular hydroxyl radical concentration measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) assay in NIH3T3. Finally, fluorescence imaging with JC-1 confirmed that the mitochondrial membranes of NIH 3T3 were well-protected during the transfection when NLS contained histidine. These experimental results confirm the hypothesis that histidine residues scavenge ROS that is generated during the transfection process, preventing the excessive damage to mitochondrial membranes, leading to reduced cytotoxicity.

VP24 matrix proteins of eight filoviruses downregulate innate immune response by inhibiting the interferon-induced pathway.

Filoviruses encode viral protein 24 (VP24) which effectively inhibit the innate immune responses in infected cells. Here we systematically analysed the effects of nine mammalian filovirus VP24 proteins on interferon (IFN)-induced immune response. We transiently expressed Ebola, Bombali, Bundibugyo, Reston, Sudan and Taï Forest ebolavirus (EBOV, BOMV, BDBV, RESTV, SUDV, TAFV, respectively), Lloviu virus (LLOV), Mengla dianlovirus (MLAV) and Marburgvirus (MARV) VP24 proteins and analysed their ability to inhibit IFN-α-induced activation of myxovirus resistance protein 1 (MxA) and interferon-induced transmembrane protein 3 (IFITM3) promoters. In addition, we analysed the expression of endogenous MxA protein in filovirus VP24-expressing cells. Eight filovirus VP24 proteins, including the VP24s of the recently discovered MLAV, BOMV and LLOV, inhibited IFN-induced MxA and IFITM3 promoter activation. MARV VP24 was the only protein with no inhibitory effect on the activation of either promoter. Endogenous MxA protein expression was impaired in cells transiently expressing VP24s with the exception of MARV VP24. We mutated nuclear localization signal (NLS) of two highly pathogenic filoviruses (EBOV and SUDV) and two putatively non-pathogenic filoviruses (BOMV and RESTV), and showed that the inhibitory effect on IFN-induced expression of MxA was dependent on functional cluster 3 of VP24 nuclear localization signal. Our findings suggest that filovirus VP24 proteins are both genetically and functionally conserved, and that VP24 proteins of most filovirus species are capable of inhibiting IFN-induced antiviral gene expression thereby efficiently downregulating the host innate immune responses.