Xianliang Ke

One-pot synthesized aptamer-functionalized CdTe:Zn2+ quantum dots for tumor-targeted fluorescence imaging in vitro and in vivo.

High quality and facile DNA functionalized quantum dots (QDs) as efficient fluorescence nanomaterials are of great significance for bioimaging both in vitro and in vivo applications. Herein, we offer a strategy to synthesize DNA-functionalized Zn(2+) doped CdTe QDs (DNA-QDs) through a facile one-pot hydrothermal route. DNA is directly attached to the surface of QDs. The as-prepared QDs exhibit small size (3.85 ± 0.53 nm), high quantum yield (up to 80.5%), and excellent photostability. In addition, the toxicity of QDs has dropped considerably because of the Zn-doping and the existence of DNA. Furthermore, DNA has been designed as an aptamer specific for mucin 1 overexpressed in many cancer cells including lung adenocarcinoma. The aptamer-functionalized Zn(2+) doped CdTe QDs (aptamer-QDs) have been successfully applied in active tumor-targeted imaging in vitro and in vivo. A universal design of DNA for synthesis of Zn(2+) doped CdTe QDs could be extended to other target sequences. Owing to the abilities of specific recognition and the simple synthesis route, the applications of QDs will potentially be extended to biosensing and bioimaging.

Human MicroRNA hsa-miR-296-5p Suppresses Enterovirus 71 Replication by Targeting the Viral Genome

ABSTRACT Enterovirus 71 (EV71) has emerged as a major cause of neurological disease following the near eradication of poliovirus. Accumulating evidence suggests that mammalian microRNAs (miRNAs), a class of noncoding RNAs of 18 to 23 nucleotides (nt) with important regulatory roles in many cellular processes, participate in host antiviral defenses. However, the roles of miRNAs in EV71 infection and pathogenesis are still unclear. Here, hsa-miR-296-5p expression was significantly increased in EV71-infected human cells. As determined by virus titration, quantitative real-time PCR (qRT-PCR), and Western blotting, overexpression of hsa-miR-296-5p inhibited, while inhibition of endogenous hsa-miR-296-5p facilitated, EV71 infection. Additionally, two potential hsa-miR-296-5p targets (nt 2115 to 2135 and nt 2896 to 2920) located in the EV71 genome (strain BrCr) were bioinformatically predicted and validated by luciferase reporter assays and Western blotting. Genomic alignment of various EV71 strains revealed synonymous mutations in hsa-miR-296-5p target sequences. Furthermore, the introduction of synonymous mutations into the EV71 BrCr genome by site-directed mutagenesis impaired the viral inhibitory effects of hsa-miR-296-5p and facilitated mutant virus infection. Meanwhile, compensatory mutations in corresponding hsa-miR-296-5p target sequences of the EV71 HeN strain (GenBank accession number JN256064) restored the inhibitory effects of the miRNA. These results indicate that hsa-miR-296-5p inhibits EV71 replication by targeting the viral genome. Our findings support the notion that cellular miRNAs can inhibit virus infection and that the virus mutates to escape suppression by cellular miRNAs.

Human Bocavirus NP1 Inhibits IFN-β Production by Blocking Association of IFN Regulatory Factor 3 with IFNB Promoter

Human bocavirus (HBoV) mainly infects young children. Although many infected children suffer from respiratory or gastroenteric tract diseases, an association between HBoV and these diseases is not definite. Because modulation of type I IFN is crucial for viruses to establish efficient replication, in this study, we tested whether HBoV modulates type I IFN production. We observed that a nearly full-length HBoV clone significantly reduced both Sendai virus (SeV)- and poly(deoxyadenylic-thymidylic) acid-induced IFN-β production. Further study showed that NP1 blocked IFN-β activation in response to SeV, poly(deoxyadenylic-thymidylic) acid, and IFN-β pathway inducers, including retinoic acid-inducible protein I, mitochondrial antiviral signaling protein, inhibitor of κB kinase ε, and TANK-binding kinase 1. In addition, NP1 interfered with IRF-3–responsive PRD(III-I) promoter activated by SeV and a constitutively active mutant of IRF-3 (IRF-3/5D). Although NP1 suppressed the IRF-3 pathway, it did not affect IRF-3 activation processes, including phosphorylation, dimerization, and nuclear translocation. Coimmunoprecipitation assays confirmed the interaction between NP1 and IRF-3. Additional deletion mutagenesis and coimmunoprecipitation assays revealed that NP1 bound to the DNA-binding domain of IRF-3, resulting in the interruption of an association between IRF-3 and IFNB promoter. Altogether, our results indicate that HBoV NP1 blocks IFN production through a unique mechanism. To our knowledge, this is the first study to investigate the modulation of innate immunity by HBoV. Our findings suggest a potential immune-evasion mechanism used by HBoV and provide a basis for better understanding HBoV pathogenesis.

Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus Infection

Utilization of quantum dots (QDs) for single virus tracking has attracted growing interest. Through modification of viral surface proteins, viruses can be labeled with various functionalized QDs and used for tracking the routes of viral infections. However, incorporation of QDs on the viral surface may affect the efficiency of viral entry and alter virus-cell interactions. Here, we describe that QDs can be encapsulated into the capsid of vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus (PTLV) in living cells without modification of the viral surface. QDs conjugated with modified genomic RNAs (gRNAs), which contain a packaging signal (Psi) sequence for viral genome encapsulation, can be packaged into virions together with the gRNAs. QD-containing PTLV demonstrated similar entry efficiency as the wild-type PTLV. After infection, QD signals entered the Rab5+ endosome and then moved to the microtubule organizing center of the infected cells in a microtubule-dependent manner. Findings in this study are consistent with previously reported infection routes of VSV and VSV-G pseudotyped lentivirus, indicating that our established QD packaging approach can be used for enveloped virus labeling and tracking.

Human Bocavirus VP2 Upregulates IFN-β Pathway by Inhibiting Ring Finger Protein 125–Mediated Ubiquitination of Retinoic Acid–Inducible Gene-I

Precise regulation of innate immunity is crucial for maintaining optimal immune responses against infections. Whereas positive regulation of IFN signaling elicits rapid type I IFNs, negative regulation is equally important in preventing the production of superfluous IFNs that can be hazardous to the host. The positive regulators of IFN pathway are known to be the main targets of viruses to antagonize the innate immune system. Whether viruses target the negative regulators of IFN pathway remains to be fully investigated. In this study, we report that the structural protein VP2 of human Bocavirus modulates IFN pathway by targeting the ring finger protein 125 (RNF125), a negative regulator of type I IFN signaling, which conjugates Lys48-linked ubiquitination to retinoic acid–inducible gene-I (RIG-I) and subsequently leads to the proteasome-dependent degradation of RIG-I. VP2 not only upregulated Sendai virus (SeV)–induced IFNB promoter activity, but also enhanced SeV-induced IFN-β production at both mRNA and protein levels. In agreement, the level of Ser396-phosphorylated IFN regulatory factor 3 stimulated by SeV was enhanced in the presence of VP2. Furthermore, VP2 was demonstrated to physically interact with RNF125, resulting in the reduction of RNF125-mediated ubiquitination and proteasome-dependent degradation of RIG-I. Additional study indicated that endogenous RIG-I degradation was decreased in VP2-expressing cells. Our study delineates a unique phenomenon for aberrant activation of IFN regulatory factor 3 pathway and may represent a new mechanism underlying viral manipulation of the host immune system.

2C proteins of enteroviruses suppress IKKβ phosphorylation by recruiting protein phosphatase 1

ABSTRACT The NF-κB signaling network, which is an ancient signaling pathway, plays a pivotal role in innate immunity and constitutes a first line of defense against invading pathogens, including viruses. However, numerous viruses possess evolved strategies to antagonize the activation of the NF-κB signaling pathway. Our previous study demonstrated that the nonstructural protein 2C of enterovirus 71 (EV71), which is the major pathogen of hand, foot, and mouth disease, inhibits tumor necrosis factor alpha (TNF-α)-mediated activation of NF-κB by suppressing IκB kinase β (IKKβ) phosphorylation. Nevertheless, the mechanism underlying the inhibition of IKKβ phosphorylation by EV71 2C remains largely elusive. We demonstrate that EV71 2C interacts with all isoforms of the protein phosphatase 1 (PP1) catalytic subunit (the PP1α, PP1β, and PP1γ isoforms) through PP1-docking motifs. EV71 2C has no influence on the subcellular localization of PP1. In addition, the PP1-binding-deficient EV71 2C mutant 3E3L nearly completely lost the ability to suppress IKKβ phosphorylation and NF-κB activation was markedly restored in the mutant, thereby indicating that PP1 binding is efficient for EV71 2C-mediated inhibition of IKKβ phosphorylation and NF-κB activation. We further demonstrate that 2C forms a complex with PP1 and IKKβ to dephosphorylate IKKβ. Notably, we reveal that other human enteroviruses, including poliovirus (PV), coxsackie A virus 16 (CVA16), and coxsackie B virus 3 (CVB3), use 2C proteins to recruit PP1, leading to the inhibition of IKKβ phosphorylation. Our findings indicate that enteroviruses exploit a novel mechanism to inhibit IKKβ phosphorylation by recruiting PP1 and IKKβ to form a complex through 2C proteins, which ultimately results in the inhibition of the NF-κB signaling pathway. IMPORTANCE The innate antiviral immunity system performs an essential function in recognizing and eliminating invading viruses. Enteroviruses include a number of important human pathogens, including poliovirus (PV), EV71, and coxsackieviruses (CVs). As 2C is the most conserved and complex nonstructural protein of enteroviruses, its biological function is largely unclear, whereas the 2A and 3C proteinases of enteroviruses are well characterized. We reveal that EV71 2C forms a complex with PP1 and IKKβ to maintain IKKβ in an unphosphorylated and inactive state, resulting in the inactivation of the TNF-α-mediated NF-κB signaling pathway. We provide evidence that the 2C proteins of the enteroviruses PV, CVA16, and CVB3 suppress IKKβ phosphorylation through the same mechanism involving PP1. We demonstrate that enteroviruses exploit a novel mechanism involving PP1 to regulate innate antiviral immunity, and our findings may be particularly important for understanding the pathogenicity of enteroviruses.

IFIT5 positively regulates NF-κB signaling through synergizing the recruitment of IκB kinase (IKK) to TGF-β-activated kinase 1 (TAK1)

Precise regulation of NF-κB signaling pathways is essential to effective host immune response. However, the specific molecular mechanism underlying NF-κB activation by different stimuli is not fully understood. Here we demonstrate that IFIT5, one of the interferon induced tetratricopeptide repeat family members, enhances IKK phosphorylation and NF-κB activation through interacting with TAK1 and IKK. Following TNF-α treatment, IFIT5 interacted with TAK1 or IKK complex and synergized the recruitment of IKK to TAK1 in a dose dependent manner. Consistent with these observations, knockdown of IFIT5 decreased the recruitment of IKK to TAK1 and markedly weakened IKK phosphorylation, further reducing the production of NF-κB target genes IL-8 and ICAM-1. Moreover, we found that IFIT5 also promoted SeV-induced IKK phosphorylation and NF-κB activation by regulating the recruitment of IKK to TAK1. Our findings identify a previously unrecognized role of IFIT5 as a positive regulator in IKK phosphorylation and NF-κB activation, highlighting that IFIT5 serves as an important mediator in innate immunity.

Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1: identification and characterization of complex dual nuclear localization signals in human bocavirus NP1.

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7-50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14-28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7-50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65

Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α–mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn’t interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis.

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

ABSTRACT Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.