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.

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.

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.

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.

Exploring a Nuclear-Selective Radioisotope Delivery System for Efficient Targeted Alpha Therapy.

Targeted alpha therapy (TAT) has garnered significant interest as an innovative cancer therapy. Owing to their high energy and short range, achieving selective α-particle accumulation in target tumor cells is crucial for obtaining high potency without adverse effects. To meet this demand, we fabricated an innovative radiolabeled antibody, specifically designed to selectively deliver 211At (α-particle emitter) to the nuclei of cancer cells. The developed 211At-labeled antibody exhibited a superior effect compared to its conventional counterparts. This study paves the way for organelle-selective drug delivery.

Subcellular distribution of bone morphogenetic protein 2-inducible kinase (BMP2K): Regulation by liquid-liquid phase separation and nucleocytoplasmic shuttling.

Bone morphogenetic protein 2 (BMP2)-inducible kinase (BMP2K) is induced by the cytokine BMP2, which is also implicated in the production of bone differentiation. In addition to regulating bone differentiation, BMP2K is implicated in a variety of cancers. Therefore, understanding the variables that determine where in the cell this kinase functions may help in understanding malignancies linked to BMP2K. However, the mechanisms regulating the subcellular localization of BMP2K are mainly unknown. By liquid-liquid phase separation (LLPS), BMP2K forms droplets in the cytoplasm, but how the droplets are regulated remains unclear. The reason why BMP2K localizes to the cytoplasm irrespective of having a nuclear localization signal (NLS) is also unknown. Here we show the element that controls BMP2K's LLPS and cytoplasmic localization. A glutamine-rich area is necessary for BMP2K phase separation, and droplet formation is controlled by hyperosmolarity. Cytoplasmic localization of BMP2K is managed by inhibition of NLS function through phosphorylation of Ser-1010 and by a newly found cytoplasmic localization region that antagonizes the NLS. These results will provide an important biochemical foundation for the advancement of BMP2K-related cell biology, structural biology, and pathophysiology.

Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation.

Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins.

A Peptide Inhibitor of the Human Cytomegalovirus Core Nuclear Egress Complex.

The replication of human cytomegalovirus (HCMV) involves a process termed nuclear egress, which enables translocation of newly formed viral capsids from the nucleus into the cytoplasm. The HCMV core nuclear egress complex (core NEC), a heterodimer of viral proteins pUL50 and pUL53, is therefore considered a promising target for new antiviral drugs. We have recently shown that a 29-mer peptide presenting an N-terminal alpha-helical hook-like segment of pUL53, through which pUL53 interacts with pUL50, binds to pUL50 with high affinity, and inhibits the pUL50-pUL53 interaction in vitro. Here, we show that this peptide is also able to interfere with HCMV infection of cells, as well as with core NEC formation in HCMV-infected cells. As the target of the peptide, i.e., the pUL50-pUL53 interaction, is localized at the inner nuclear membrane of the cell, the peptide had to be equipped with translocation moieties that facilitate peptide uptake into the cell and the nucleus, respectively. For the resulting fusion peptide (NLS-CPP-Hook), specific cellular and nuclear uptake into HFF cells, as well as inhibition of infection with HCMV, could be demonstrated, further substantiating the HCMV core NEC as a potential antiviral target.

Mitotic genome bookmarking by nuclear receptor VDR advocates transmission of cellular transcriptional memory to progeny cells.

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as 'genome bookmarking factors' and the phenomenon is termed 'genome bookmarking'. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full 'DNA binding domain (DBD)' present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1(Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells.

Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9.

The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.

A Bivalent Supramolecular GCP Ligand Enables Blocking of the Taspase1/Importin α Interaction.

Taspase1 is a unique protease not only pivotal for embryonic development but also implicated in leukemia as well as solid tumors. As such, it is a promising target in cancer therapy, although only a limited number of Taspase1 inhibitors lacking general applicability are currently available. Here we present a bivalent guanidiniocarbonyl-pyrrole (GCP)-containing supramolecular ligand that is capable of disrupting the essential interaction between Taspase1 and its cognate import receptor Importin α in a concentration-dependent manner in vitro with an IC50 of 35 µM. Here, size of the bivalent vs the monovalent construct as well as its derivation with an aromatic cbz-group arose as critical determinants for efficient interference of 2GC. This was also evident when we investigated the effects in different tumor cell lines, resulting in comparable EC50 values (∼40-70 µM). Of note, in higher concentrations, 2GC also interfered with Taspase1's proteolytic activity. We thus believe to set the stage for a novel class of Taspase1 inhibitors targeting a pivotal protein-protein interaction prerequisite for its cancer-associated proteolytic function.

Molecular Coevolution of Nuclear and Nucleolar Localization Signals inside the Basic Domain of HIV-1 Tat.

During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses. IMPORTANCE Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (49RKKRRQRRR57) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.

The Anti-tumor Nano-drug Carrier System Modified by theNuclear Localization Signal Peptide / 中国生物化学与分子生物学报

Nano-drug carrier systems, as the controllable and targeting tool to deliver drugs, can effectively improve the drug bioavailability, enhance their therapeutic outcomes and reduce side effects, mainly through protecting drugs from rapid enzymatic degradation and blood clearance and ensuring them to be delivered to the targeting sites. The nano-drug carrier system owns broad application prospects in the biomedical field and attracts increasing attention in both functional materials and anti-tumor research. Recently, functional surface modification with functional biomolecules to improve the biocompatibility and drug bioactivity is a hot topic in nano medicine research. The nucleus is the main site of action for manyanti-tumor substances. And nuclear localization signal (NLS) peptides, as a type of functional peptides with nuclear-targeting activity, can penetrate through biological membranes and target the nucleus and is considered to be a universal tool for constructing nano-drug carrier systems. The use of NLS peptides to construct a functionalized nano-drug carrier system with nuclear targeting ability has important application values in the field of anti-tumor therapy. Although the synthesis process of nuclear-targeted functionalized nano-drug carrier system has been developed, due to the high preparation cost and complex synthesis process, there is still a long research process in the successful translation of nuclear-targeted nanocarriers from the experimental stage to the clinical stage. This review mainly focuses on the composition and construction of the nuclear-targeted functionalized nano-drug carrier system, analyzes its nuclear entry methods and conditions, and prospects the development of the anti-tumor nano-drug carrier system in the future based on the current challenges.

Jacob, a Synapto-Nuclear Protein Messenger Linking N-methyl-D-aspartate Receptor Activation to Nuclear Gene Expression.

Pyramidal neurons exhibit a complex dendritic tree that is decorated by a huge number of spine synapses receiving excitatory input. Synaptic signals not only act locally but are also conveyed to the nucleus of the postsynaptic neuron to regulate gene expression. This raises the question of how the spatio-temporal integration of synaptic inputs is accomplished at the genomic level and which molecular mechanisms are involved. Protein transport from synapse to nucleus has been shown in several studies and has the potential to encode synaptic signals at the site of origin and decode them in the nucleus. In this review, we summarize the knowledge about the properties of the synapto-nuclear messenger protein Jacob with special emphasis on a putative role in hippocampal neuronal plasticity. We will elaborate on the interactome of Jacob, the signals that control synapto-nuclear trafficking, the mechanisms of transport, and the potential nuclear function. In addition, we will address the organization of the Jacob/NSMF gene, its origin and we will summarize the evidence for the existence of splice isoforms and their expression pattern.

Genetic Patterns Found in the Nuclear Localization Signals (NLSs) Associated with EBV-1 and EBV-2 Provide New Insights into Their Contribution to Different Cell-Type Specificities.

The Epstein-Barr virus (EBV) is a globally dispersed pathogen involved in several human cancers of B-cell and non-B-cell origin. EBV has been classified into EBV-1 and EBV-2, which have differences in their transformative ability. EBV-1 can transform B-cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein is a transcriptional regulator of virus and host cell genes, and is required in order to transform B-cells. EBNA3A has six peptide motifs called nuclear localization signals (NLSs) that ensure nucleocytoplasmic protein trafficking. The presence of multiple NLSs has been suggested to enhance EBNA3 function or different specificities in different cell types. However, studies about the NLS variability associated with EBV types are scarce. Based on a systematic sequence analysis considering more than a thousand EBNA3A sequences of EBV from different human clinical manifestations and geographic locations, we found differences in NLSs' nucleotide structures among EBV types. Compared with the EBNA3A EBV-1, EBNA3A EBV-2 has two of the six NLSs altered, and these mutations were possibly acquired by recombination. These genetic patterns in the NLSs associated with EBV-1 and EBV-2 provide new information about the traits of EBNA3A in EBV biology.

Role of Intracellular Distribution of Feline and Bovine SAMHD1 Proteins in Lentiviral Restriction.

Human SAMHD1 (hSAM) restricts lentiviruses at the reverse transcription step through its dNTP triphosphohydrolase (dNTPase) activity. Besides humans, several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1. However, the intracellular distribution of feline and bovine SAMHD1 (fSAM and bSAM) and its significance in their lentiviral restriction function is not known. Here, we demonstrated that fSAM and bSAM were both predominantly localized to the nucleus and nuclear localization signal (11KRPR14)-deleted fSAM and bSAM relocalized to the cytoplasm. Both cytoplasmic fSAM and bSAM retained the antiviral function against different lentiviruses and cytoplasmic fSAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type (WT) protein as cytoplasmic hSAM. Further investigation revealed that cytoplasmic fSAM was resistant to Vpx-induced degradation like cytoplasmic hSAM, while cytoplasmic bSAM was not, but they all demonstrated the same in vitro dNTPase activity and all could interact with Vpx as their WT proteins, indicating that cytoplasmic hSAM and fSAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation. Our results suggested that fSAM- and bSAM-mediated lentiviral restriction does not require their nuclear localization and that fSAM shares more common features with hSAM. These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.

Nonviral gene delivery using PAMAM dendrimer conjugated with the nuclear localization signal peptide derived from human papillomavirus type 11 E2 protein.

Polyamidoamine (PAMAM) dendrimers are biocompatible polymers utilized in multiple biomedical applications including tissue engineering, medical diagnosis, drug and gene delivery systems, and biosensors. Normally, high-generation PAMAM dendrimers are advantageous for use in gene therapy research because they have a relatively high transfection efficiency. A high-generation PAMAM dendrimer has a high charge density, which induces greater damage to the membranous organelles than that induced by a low-generation PAMAM dendrimer. In this study, we added NLS sequences derived from the human papillomavirus (HPV) type 11 E2 protein to the low-generation PAMAM generation 2 (PAMAM G2) dendrimer and simultaneously introduced histidine residues to reduce cytotoxicity. RKRARH-PAMAM G2 showed similar and high transfection efficiencies in Neuro-2A and NIH3T3 cell lines and relatively low cytotoxicities relative to that of polyethylenimine 25 kDa (PEI 25 kDa).

The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7).

BACKGROUND: Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far METHODS: An unsupervised in silico approach was used, followed by experimental validation. RESULTS: We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. CONCLUSIONS: The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for ß-karyopherin family members. GENERAL SIGNIFICANCE: Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.

Systematic analysis of nuclear localization of Autographa californica multiple nucleopolyhedrovirus proteins.

Baculoviruses are large DNA viruses that replicate within the nucleus of infected host cells. Therefore, many viral proteins must gain access to the nucleus for efficient viral genome replication, gene transcription and virion assembly. To date, the global protein localization pattern of baculoviral proteins is unknown. In this study, we systematically analysed the nuclear localization of 154 ORFs encoded by the prototypic baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), either during transient expression or with super-infection of the virus. By transient expression of vectors containing egfp-fused ORFs, we found that in the absence of virus infection, 25 viral proteins were localized in the nucleus. Most of these, which we called 'auto-nuclear localization' proteins, are related to virus replication, transcription or virion structure, and 20 of them contain predicted classical nuclear localization signal. Upon virus infection, 11 proteins, which originally localized in the cytoplasm or both cytoplasm and nucleus in the transfection assays, were completely translocated into the nucleus, suggesting that their nuclear import is facilitated by other viral or host proteins. Further co-transfection experiments identified that four of the 11 proteins, including P143, P33, AC73 and AC114, were imported into the nucleus with the assistance of the auto-nuclear localization proteins LEF-3 (for P143), TLP (for P33) and VP80 (for both AC73 and AC114). This study presents the first global nuclear localization profile of AcMNPV proteins and provides useful information for further elucidation of the mechanisms of baculovirus nuclear entry and gene functions.

Nuclear localization of Zika virus NS5 contributes to suppression of type I interferon production and response.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which caused an unprecedented epidemic in Latin America. Among all viral non-structural proteins in flavivirus, NS5 is the most highly conserved and has multiple crucial functions, including participating in viral RNA replication and suppressing host innate immunity. Although ZIKV NS5 prominently localizes in the nucleus during infection, its specific nuclear localization signal (NLS), and its role in viral replication and pathogenesis remain controversial. Here, we identified aa 11-90 and aa 370-406 regions that contain NLSs, which are critical for nuclear localization of ZIKV NS5. Further experiments demonstrated that nuclear localization of ZIKV NS5 predominantly participates in suppression of interferon regulatory factor 3 (IRF3)-mediated activation of type I IFN (IFN-I) transcription and inhibition of IFN-I downstream response independent of its effect on signal transducers and activators of transcription 2 (STAT2) degradation. These results suggest that subcellular localization of NS5 is important for its function on innate immune suppression, which provides new insight into ZIKV pathogenesis.