Bulleyaconitine A is a sensitive substrate and competitive inhibitor of CYP3A4: One of the possible explanations for clinical adverse reactions.

Bulleyaconitine A (BLA), a toxic Aconitum alkaloid, is a potent analgesic that is clinically applied to treat rheumatoid arthritis, osteoarthritis and lumbosacral pain. BLA-related adverse reactions occur frequently, but whether the underlying mechanism is related to its metabolic interplay with drug-metabolizing enzymes remains unclear. This study aimed to elucidate the metabolic characteristics of BLA and its affinity action and mechanism to drug-metabolizing enzymes to reveal whether BLA-related adverse reactions are modulated by enzymes. After incubation with human liver microsomes and recombinant human cytochrome P450 enzymes, we found that BLA was predominantly metabolized by CYP3A, in which CYP3A4 had an almost absolute advantage. In vitro, the CYP3A4 inhibitor ketoconazole noticeably suppressed the metabolism of BLA. In vivo, the AUC0-∞ values, cardiotoxicity and neurotoxicity of BLA in Cyp3a-inhibited mice were all obviously enhanced (P < 0.05) compared to those in normal mice. In the enzyme kinetics study, BLA was found to be a sensitive substrate of CYP3A4, and its characteristics were consistent with substrate inhibition (Km = 39.36 ± 10.47 µmol/L, Ks = 83.42 ± 19.65 µmol/L). BLA was further identified to be a competitive inhibitor of CYP3A4 with Ki = 53.64 µmol/L, since the intrinsic clearance (CLint) of midazolam, a selective CYP3A4 substrate, decreased significantly (P < 0.05) when incubated with BLA together in mouse liver microsomes. Overall, BLA is a sensitive substrate and competitive inhibitor of CYP3A4, and clinical adverse reactions of BLA may mechanistically related to the CYP3A4-mediated drug-drug interactions.

The Molecular Mechanism of Herpes Simplex Virus 1 UL31 in Antagonizing the Activity of IFN-ß.

Virus infection triggers intricate signal cascade reactions to activate the host innate immunity, which leads to the production of type I interferon (IFN-I). Herpes simplex virus 1 (HSV-1), a human-restricted pathogen, is capable of encoding over 80 viral proteins, and several of them are involved in immune evasion to resist the host antiviral response through the IFN-I signaling pathway. Here, we determined that HSV-1 UL31, which is associated with nuclear matrix and is essential for the formation of viral nuclear egress complex, could inhibit retinoic acid-inducible gene I (RIG-I)-like receptor pathway-mediated interferon beta (IFN-ß)-luciferase (Luc) and (PRDIII-I)4-Luc (an expression plasmid of IFN-ß positive regulatory elements III and I) promoter activation, as well as the mRNA transcription of IFN-ß and downstream interferon-stimulated genes (ISGs), such as ISG15, ISG54, ISG56, etc., to promote viral infection. UL31 was shown to restrain IFN-ß activation at the interferon regulatory factor 3 (IRF3)/IRF7 level. Mechanically, UL31 was demonstrated to interact with TANK binding kinase 1 (TBK1), inducible IκB kinase (IKKi), and IRF3 to impede the formation of the IKKi-IRF3 complex but not the formation of the IRF7-related complex. UL31 could constrain the dimerization and nuclear translocation of IRF3. Although UL31 was associated with the CREB binding protein (CBP)/p300 coactivators, it could not efficiently hamper the formation of the CBP/p300-IRF3 complex. In addition, UL31 could facilitate the degradation of IKKi and IRF3 by mediating their K48-linked polyubiquitination. Taken together, these results illustrated that UL31 was able to suppress IFN-ß activity by inhibiting the activation of IKKi and IRF3, which may contribute to the knowledge of a new immune evasion mechanism during HSV-1 infection. IMPORTANCE The innate immune system is the first line of host defense against the invasion of pathogens. Among its mechanisms, IFN-I is an essential cytokine in the antiviral response, which can help the host eliminate a virus. HSV-1 is a double-stranded DNA virus that can cause herpes and establish a lifelong latent infection, due to its possession of multiple mechanisms to escape host innate immunity. In this study, we illustrate for the first time that the HSV-1-encoded UL31 protein has a negative regulatory effect on IFN-ß production by blocking the dimerization and nuclear translocation of IRF3, as well as promoting the K48-linked polyubiquitination and degradation of both IKKi and IRF3. This study may be helpful for fully understanding the pathogenesis of HSV-1.

Plasmonic gold nanostructures for biosensing and bioimaging.

As one kind of noble metal nanostructures, the plasmonic gold nanostructures possess unique optical properties as well as good biocompatibility, satisfactory stability, and multiplex functionality. These distinctive advantages make the plasmonic gold nanostructures an ideal medium in developing methods for biosensing and bioimaging. In this review, the optical properties of the plasmonic gold nanostructures were firstly introduced, and then biosensing in vitro based on localized surface plasmon resonance, Rayleigh scattering, surface-enhanced fluorescence, and Raman scattering were summarized. Subsequently, application of the plasmonic gold nanostructures for in vivo bioimaging based on scattering, photothermal, and photoacoustic techniques  has been also briefly covered. At last, conclusions of the selected examples are presented and an outlook of this research topic is given.

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species.

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Mediate the Disposition of Leonurine-10-O-ß-glucuronide.

BACKGROUND: Leonurine (Leo), a promising antilipemic agent that has been approved for clinical trials, is extensively metabolized into bioactive Leonurine-10-O-ß-glucuronide (L-10-G) vivo. OBJECTIVE: To explore the effects of breast cancer resistance protein (Bcrp) and multidrug resistance protein 2 (Mrp2) on the disposition of L-10-G. METHODS: The pharmacokinetics, tissue distribution and intestinal perfusion of Leo were studied by using efflux transporter gene knockout mouse models. The enzyme kinetics via liver and intestinal microsomes were also examined. RESULTS: After intravenous injection with Leo, the AUC0-∞ values of L-10-G in Bcrp1-/- and Mrp2-/- mice were 1.55-fold and 16.80-fold higher, respectively, than those in wild-type FVB mice (P < 0.05). After oral administration, the AUC0-∞ value of L-10-G showed a 2.82-fold increase in Mrp2-/- mice compared with wild-type FVB mice (P < 0.05). After gavage with Leo for 10 and 25 min, the bile accumulation of L-10-G in Mrp2-/- mice was 3-fold and 22-fold lower, respectively, than that in wild-type FVB mice (P < 0.05). Besides, the intestinal excreted amount of L-10-G showed 2.22-fold and 2.68-fold decrease in Bcrp1-/- and Mrp2-/- mice, respectively, compared with that in wild-type FVB mice (P < 0.05). The clearance of L-10-G decreased in liver microsomes and increased in intestinal microsomes of Bcrp1-/- and Mrp2-/- mice compared to the wild-type FVB mice (P < 0.05). CONCLUSION: Both Bcrp and Mrp2 are involved in the disposition of L-10-G, and Mrp2 exhibits a superior influence.

Mdr1a, Bcrp and Mrp2 regulate the efficacy and toxicity of mesaconitine and hypaconitine by altering their tissue accumulation and in vivo residence.

Mesaconitine (MA) and hypaconitine (HA) are the main bioactive/toxic alkaloids of Aconitum carmichaelii Debx, and MDR1, BCRP and MRP2 are involved in their efflux in vitro. This study aimed to explore the effects of Mdr1a, Bcrp and Mrp2 on the efficacy/toxicity of MA and HA by using efflux transporter gene knockout mouse models. The analgesic and anti-inflammatory effects, neurotoxicity/cardiotoxicity, and pharmacokinetic profiles of MA and HA were studied. Compared to wild-type mice, the analgesic effects of MA or HA were significantly enhanced in Mdr1a--/-, Bcrp1-/- and Mrp2-/- mice, and the anti-inflammatory effects notably increased in Bcrp1-/- and Mrp2-/- mice. Compared to wild-type mice, Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice suffered from severe karyopyknosis and edema in the brain after MA or HA treatment. Meanwhile, significant arrhythmia appeared, and the heart rate and RR-interval were greatly altered in Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice. Additionally, obvious disorder of cardiomyocytes were observed, and the CK and cTnT (indicators of heart injury) levels were greatly enhanced in efflux transporter gene knockout mice. The brain levels of MA and HA were markedly increased in Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice, and the heart levels of MA and HA enhanced greatly in Mdr1a-/- mice. The MRT0-t values of MA and HA were remarkably enhanced in most efflux transporter gene knockout mice. In conclusion, Mdr1a, Bcrp and Mrp2 were all involved in regulating the efficacy/toxicity of MA and HA by altering their tissue accumulation and in vivo residence. Among the three efflux transporters, Mdr1a had a superior regulatory effect.

Herpes Simplex Virus 1 UL2 Inhibits the TNF-α-Mediated NF-κB Activity by Interacting With p65/p50.

Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that encodes at least 80 viral proteins, many of which are involved in the virus-host interaction and are beneficial to the viral survival and reproduction. However, the biological functions of some HSV-1-encoded proteins are not fully understood. Nuclear factor κB (NF-κB) activation is the major antiviral innate response, which can be triggered by various signals induced by cellular receptors from different pathways. Here, we demonstrated that HSV-1 UL2 protein could antagonize the tumor necrosis factor α (TNF-α)-mediated NF-κB activation. Co-immunoprecipitation assays showed that UL2 could interact with the NF-κB subunits p65 and p50, which also revealed the region of amino acids 9 to 17 of UL2 could suppress the NF-κB activation and interact with p65 and p50, and UL2 bound to the immunoglobulin-like plexin transcription factor functional domain of p65. However, UL2 did not affect the formation of p65/p50 dimerization and their nuclear localizations. Yet, UL2 was demonstrated to inhibit the NF-κB activity by attenuating TNF-α-induced p65 phosphorylation at Ser536 and therefore decreasing the expression of downstream inflammatory chemokine interleukin 8. Taken together, the attenuation of NF-κB activation by UL2 may contribute to the escape of host's antiviral innate immunity for HSV-1 during its infection.

Molecular anatomy of the subcellular localization and nuclear import mechanism of herpes simplex virus 1 UL6.

As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production.

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12RRR14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Epstein-Barr Virus Early Protein BFRF1 Suppresses IFN-ß Activity by Inhibiting the Activation of IRF3.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis that is closely associated with several human malignant diseases, while type I interferon (IFN-I) plays an important role against EBV infection. As we all know, EBV can encode some proteins to inhibit the production of IFN-I, but it's not clear whether other proteins also take part in this progress. EBV early lytic protein BFRF1 is shown to be involved in viral maturation, however, whether BFRF1 participates in the host innate immune response is still not well known. In this study, we found BFRF1 could down-regulate sendai virus-induced IFN-ß promoter activity and mRNA expression of IFN-ß and ISG54 during BFRF1 plasmid transfection and EBV lytic infection, but BFRF1 could not affect the promoter activity of NF-κB or IRF7. Specifically, BFRF1 could co-localize and interact with IKKi. Although BFRF1 did not interfere the interaction between IKKi and IRF3, it could block the kinase activity of IKKi, which finally inhibited the phosphorylation, dimerization, and nuclear translocation of IRF3. Taken together, BFRF1 may play a critical role in disrupting the host innate immunity by suppressing IFN-ß activity during EBV lytic cycle.

Intracellular distribution of pseudorabies virus UL2 and detection of its nuclear import mechanism.

Pseudorabies virus (PRV) UL2 (pUL2) is a multifunctional protein, which is homologous with herpes simplex virus 1 early protein UL2 (hUL2) and crucial for the viral propagation. Yet, how pUL2 executes its roles in the viral life cycle remain inadequately understood. In order to uncover its effect on the procedure of PRV infection, investigation was performed to examine the subcellular distribution of pUL2 and establish its trafficking mechanism. In the present study, enhanced yellow fluorescent protein or Myc tag fused pUL2 was transiently overexpressed in transfected cells and exhibited an absolutely nuclear accumulation without the existence of other PRV proteins. Additionally, the nuclear trafficking of pUL2 was proved to rely on Ran-, transportin-1, importin ß1, importin α1, α3 and α5. Accordingly, these data will benefit the knowledge of pUL2-mediated biological effects in PRV infection cycle.

Microfluidic chip electrophoresis for biochemical analysis.

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics-based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.

Expression, Purification, and Antiserum Production of the Truncated UL31 Protein of Herpes Simplex Virus 1.

BACKGROUND: The UL31 protein of herpes simplex virus 1 (HSV-1) plays an important role in the HSV-1 replication, however, its pinpoint functions in the life cycle of the virus have yet to be adequately elucidated. OBJECTIVES: An antiserum specific for detecting HSV-1 UL31 was prepared as the foundation for future research on the role of UL31 in the course of HSV-1 infection. MATERIALS AND METHODS: Recombinant protein of UL31 was expressed in Escherichia coli, which was then purified and employed to raise the level of antiserum in mice. Subsequently, western blot and immunofluorescence assay (IFA) were utilized to detect the specific antiserum. RESULTS: The recombinant UL31 protein consisting of N-terminal 27 aa of UL31 was fused to EYFP and His-tag. It was expressed, purified, and applied to the preparation of the antiserum. Western blot analysis and IFA demonstrated that this antiserum could detect both the recombinant UL31 and the native UL31. CONCLUSIONS: Our results manifest that this antiserum could be conducive to further investigations concerning the roles of UL31 in the HSV-1 infection.

Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF-κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF-α-induced NF-κB activity. BGLF2 was shown to interplay with the NF-κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65-p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF-κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF-κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.-Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Identification of the molecular determinants for nuclear import of PRV EP0.

Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, ß1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.

Single-cell Analysis with Microfluidic Devices.

A microfluidic device as a pivotal research tool in chemistry and life science is now widely recognized. Indeed, microfluidic techniques have made significant advancements in fundamental research, such as the inherent heterogeneity of single-cells studies in cell populations, which would be helpful in understanding cellular molecular mechanisms and clinical diagnosis of major diseases. Single-cell analyses on microdevices have shown great potential for precise fluid control, cell manipulation, and signal output with rapid and high throughput. Moreover, miniaturized devices also have open functions such as integrating with traditional detection methods, for example, optical, electrochemical or mass spectrometry for single-cell analysis. In this review, we summarized recent advances of single-cell analysis based on various microfluidic approaches from different dimensions, such as in vitro, ex vivo, and in vivo analysis of single cells.

Coordination-induced structural changes of DNA-based optical and electrochemical sensors for metal ions detection.

Metal ions play a critical role in human health and abnormal levels are closely related to various diseases. Therefore, the detection of metal ions with high selectivity, sensitivity and accuracy is particularly important. This article highlights and comments on the coordination-induced structural changes of DNA-based optical, electrochemical and optical-electrochemical-combined sensors for metal ions detection. Challenges and potential solutions of DNA-based sensors for the simultaneous detection of multiple metal ions are also discussed for further development and exploitation.

The Interaction Mechanism Between Herpes Simplex Virus 1 Glycoprotein D and Host Antiviral Protein Viperin.

Viperin is an interferon-inducible protein that responsible for a variety of antiviral responses to different viruses. Our previous study has shown that the ribonuclease UL41 of herpes simplex virus 1 (HSV-1) can degrade the mRNA of viperin to promote HSV-1 replication. However, it is not clear whether other HSV-1 encoded proteins can regulate the function of viperin. Here, one novel viperin associated protein, glycoprotein D (gD), was identified. To verify the interaction between gD and viperin, gD and viperin expression plasmids were firstly co-transfected into COS-7 cells, and fluorescence microscope showed they co-localized at the perinuclear region, then this potential interaction was confirmed by co-immunoprecipitation (Co-IP) assays. Moreover, confocal microscopy demonstrated that gD and viperin co-localized at the Golgi body and lipid droplets. Furthermore, dual-luciferase reporter and Co-IP assays showed gD and viperin interaction leaded to the increase of IRF7-mediated IFN-ß expression through promoting viperin and IRAK1 interaction and facilitating K63-linked IRAK1 polyubiquitination. Nevertheless, gD inhibited TRAF6-induced NF-κB activity by decreasing the interaction of viperin and TRAF6. In addition, gD restrained viperin-mediated interaction between IRAK1 and TRAF6. Eventually, gD and viperin interaction was corroborated to significantly inhibit the proliferation of HSV-1. Taken together, this study would open up new avenues toward delineating the function and physiological significance of gD and viperin during HSV-1 replication cycle.

Simultaneous detection of telomerase and miRNA with graphene oxide-based fluorescent aptasensor in living cells and tissue samples.

Telomerase and microRNAs (miRNAs) as important biomarkers are closely related to cancers. Simultaneous detection of telomerase activity and miRNAs would be beneficial to improve the specificity and reliability. Here, we establish a telomerase and miRNA-21 (miR-21) simultaneous sensing platform with graphene oxide-based fluorescent aptasensors (GOFA) including graphene oxide (GO), template strand (TS) primer and fluorophore-labeled telomerase/miR-21 oligonucleotides. Owing to π-π stacking interaction, TS primer and telomerase/miR-21 probes would be loaded onto GO, resulting in fluorescence quenching. However, in the presence of the telomerase or miR-21, the double-stranded oligonucleotides would be away from the GO surface attribute to the hybridization between the extended TS primers and telomerase probe as well as miR-21 and miR-21 probe, leading to obvious fluorescence recovery. We found that GOFA could simultaneously detect telomerase activity and miR-21 with low background signal, high sensitivity and simplified operation. Moreover, GOFA could be used for accurately detecting telomerase activity and miRNA in living cells and cancer patient tissue sample. This sensing platform shows great potential in improving the accuracy in clinical diagnosis of cancer.

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2.

BACKGROUND/AIMS: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. METHODS: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. RESULTS: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, 22RRLMHPHHRNYTASKASAH40, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin ß1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. CONCLUSION: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.