Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

Presence of a long nuclear-localization signal sequence in homeodomain transcription factor Nkx 1.2

Homeodomains,a 60-amino acid sequence encoded by 180 nucleotides,are highly conserved DNA-binding motifs that are present in a variety of transcription factors in species ranging from yeast to humans.The NKX proteins belong to the homeodomain(HD)-containing transcription factor family.They play vital roles in the regulation of morphogenesis.NKX1-2 is one member of the NKX subfamily.At present,information about its nuclear localization signal(NLS)sequence is limited.We studied the NLS sequence of zebrafish Nkx1.2 by introducing sequence changes such as deletion,mutation,and truncation,and identified an NLS motif(QNRRTKWKKQ)that is localized at the C-terminus of the homeodomain.Moreover,the deletion of two amino acid residues(RR)in this NLS motif prevents Nkx1.2 from entering the nucleus,indicating that the two amino acids are essential for Nkx1.2 nuclear localization.However,the NLS motif alone is unable to target cytoplasmic protein glutathione S-transferase(GST)to the nucleus.An intact homeodomain is necessary for mediating the complete nuclear transport of cytoplasmic protein.Unlike most nuclear import proteins with short NLS sequences,a long NLS is present in zebrafish Nkx1.2.We also demonstrated that the sequences of homeodomain of NKX1.2 are well conserved among different species.This study is informative to verify the function of the NKX1.2 protein.

Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis

In this study, 107 types of human papillomavirus （HPV） L1 protein sequences were obtained from available databases, and the nuclear localization signals （NLSs） of these HPV L1 proteins were analyzed and predicted by bioinformatic analysis. Out of the 107 types, the NLSs of 39 types were predicted by PredictNLS software （35 types of bipartite NLSs and 4 types of monopartite NLSs）. The NLSs of the remaining HPV types were predicted according to the characteristics and the homology of the already predicted NLSs as well as the general rule of NLSs. According to the result, the NLSs of 107 types of HPV L1 proteins were classified into 15 categories. The different types of HPV L1 proteins in the same NLS category could share the similar or the same nucleocytoplasmic transport pathway. They might be used as the same target to prevent and treat different types of HPV infection. The results also showed that bioinformatic technology could be used to analyze and predict NLSs of proteins.

A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers

Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal（s） （NLS） has not been identified. KLF8 shares with other KLFs monopartite NLSs （mNLS） and C2H2 zinc fingers （ZFs）, both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn^2＋-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of 〉1.5 ZFs from Cterminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid （aa） 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.

Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase

AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.METHODS: Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.RESULTS: We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection.CONCLUSION: Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

MOLECULAR CLONING, EXPRESSION AND SUBCELLULAR LOCALIZATION OF HUMAN OCT-4 PROTEIN

Objective To clone, express, purify human Oct-4 and detect its subcellular localization. Methods Human Oct-4 cDNA was amplified by RT-PCR strategy. Oct-4 protein was induced and expressed in BL21(DE3) strain. Furthermore, the protein was purified with Ni-NTA resin. Subsequently, site-directed mutagen-esis of Oct-4 (aa 236 - 240) was introduced. Finally, the subcellular localization of wide type Oct-4 and mutant Oct-4 was examined by immunofluorescent cytochemistry staining and confocal laser scanning microscope analysis. Results The full length cDNA of human Oct-4 was 1083 bp. Human Oct-4 encoded a 55 kd protein by prokaryotic vector in E coli. Compared with pure nuclear localization of wide type Oct-4, mutant Oct-4 was mostly enriched in the cytoplasm. Conclusion The cloning, expression and investigation of subcellular localization of human Oct-4 are basis of studying its biological function.

Intracellular transport of hepatitis B virus

For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes （NPC） is mediated by the nuclear transport receptors importin α andβ. Importin α binds to the C-terminus of the capsid protein that comprises a nuclear localization signal （NLS）. The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin α interacts with importin β that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.

A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana

PAR1 is an atypical basic-helix-loop-helix （bHLH） protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor. Consistently with this function, PAR1 has to be in the nucleus to display biological activity. Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus. However, truncated forms of PAR1 lacking this region still display biological activity, implying that PAR1 has additional mechanisms to localize into the nucleus. In this work, we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins, which led us to suggest that PAR1 contains a non-canonical nuclear localization signal （NLS） in the N-terminal region. By overexpressing truncated and mutated derivatives of PAR1, we have also investigated the importance of other regions of PAR1, such as the acidic and the extended HLH dimerization domains, for its nuclear localization. We found that, in the absence of the N-terminal region, a functional HLH domain is required for nuclear localization. Our results suggest the existence of a dual mechanism for PAR1 nuclear localization： （1） one mediated by the N-terminal non-consensus NLS and （2） a second one that involves interaction with other proteins via the dimerization domain,

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Identification of karyopherin-alpha 2 as an Oct4 associated protein

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem （ES） cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain （containing POU domain and homeodomain） of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopberin-alpha 2 （KPNA-2）. KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal （NLS） of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

A novel hierarchical targeting and controllable smart nanoparticles for enhanced in situ nuclear photodynamic therapy

Photodynamic therapy(PDT)is a promising and non-invasive treatment for various cancers.Although nuclear PDT has considerable therapeutic prospects,it is still hindered by the non-specific recognition of tumor tissues or the degradation of nuclear targeting cationic groups by enzymes in the blood.Herein,a hierarchical targeted and controlled release strategy is proposed by using folate-modified poly-β-cyclodextrin(poly-β-CD)as a nano-carrier for loading nuclear localization signals(NLSs)-conjugated photosensitizer PAP(PAP=pyropheophorbide a-PAAKRVKLD).Excitingly,the obtained FA-CD@PAP(FA=folic acid)and nanoparticles(NPs)can specifically recognize tumor cells overexpressing folate receptors(FR)to remarkedly enhance the intracellular accumulation.Furthermore,the encapsulated PAP can be released under acidic conditions to realize precise nuclear localization.The reactive oxygen species(ROS)generated by the intranuclear-accumulated PAP upon irradiation can oxidize and destroy DNA chains or DNA repair enzymes instantaneously,which can directly induce cell death.As a result,FA-CD@PAP NPs exhibit excellent tumor regression and negligible side effects.This work provides an intelligent nuclear-targeted delivery strategy for in situ nuclear PDT with extremely prominent efficacy and high biological safety.

The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Pall Complex in Arabidopsis

SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.

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

Human SAMHD1(h SAM)restricts lentiviruses at the reverse transcription step through its d NTP triphosphohydrolase(d NTPase)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(f SAM and b SAM)and its significance in their lentiviral restriction function is not known.Here,we demonstrated that f SAM and b SAM were both predominantly localized to the nucleus and nuclear localization signal(11KRPR14)-deleted f SAM and b SAM relocalized to the cytoplasm.Both cytoplasmic f SAM and b SAM retained the antiviral function against different lentiviruses and cytoplasmic f SAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type(WT)protein as cytoplasmic h SAM.Further investigation revealed that cytoplasmic f SAM was resistant to Vpx-induced degradation like cytoplasmic h SAM,while cytoplasmic b SAM was not,but they all demonstrated the same in vitro d NTPase activity and all could interact with Vpx as their WT proteins,indicating that cytoplasmic h SAM and f SAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation.Our results suggested that f SAM-and b SAM-mediated lentiviral restriction does not require their nuclear localization and that f SAM shares more common features with h SAM.These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.