Xiaowei Zhang

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.

Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions

Monitoring protein-protein interactions (PPIs) in live subjects is critical for understanding these fundamental biological processes. Bimolecular fluorescence complementation (BiFC) provides a good technique for imaging PPIs; however, a BiFC system with a long wavelength remains to be pursued for inxa0vivo imaging. Here, we conducted systematic screening of split reporters from a bacterial phytochrome-based, near-infrared fluorescent protein (iRFP). Several new near-infrared phytochrome BiFC systems were built based on selected split sites including the amino acids residues 97/98, 99/100, 122/123, and 123/124. These new near-infrared BiFC systems from a bacterial phytochrome were verified as powerful tools for imaging PPIs under physiological conditions in live cells and in live mice. The interaction between HIV-1 integrase (IN) and cellular cofactor protein Lens epithelium-derived growth factor (LEDGF/p75) was visualized in live cells using the newly constructed iRFP BiFC system because of its important roles in HIV-1 integration and replication. Because the HIV IN-LEDGF/p75 interaction is an attractive anti-HIV target, drug evaluation assays to inhibit the HIV IN-LEDGF/p75 interaction were also performed using the newly constructed BiFC system. The results showed that compound 6 and carbidopa inhibit the HIV IN-LEDGF/p75 interaction in a dose-dependent manner under physiological conditions in the BiFC assays. This study provides novel near-infrared BiFC systems for imaging protein interactions under physiological conditions and provides guidance for splitting other bacterial phytochrome-like proteins to construct BiFC systems. The study also provides a new method for drug evaluation in live cells based on iRFP BiFC systems and supplies some new information regarding candidate drugs for anti-HIV therapies.

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.

In vivo imaging of protein–protein and RNA–protein interactions using novel far-red fluorescence complementation systems

Imaging of protein–protein and RNA–protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the ‘tissue optical window’ for imaging of protein–protein and RNA–protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein–protein and mRNA–protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3′long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA–protein and protein–protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.

Human Astrocytic Cells Support Persistent Coxsackievirus B3 Infection

ABSTRACT Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.

Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages

Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and observed that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-positive endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Additionally, we observed that a dynamic actin cytoskeleton is critical for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.

Real-Time Imaging of Single HIV-1 Disassembly with Multicolor Viral Particles

Viral disassembly is poorly understood and related to the infection mechanism. However, directly observing the process in living cells remains technically challenging. In this study, the genome RNA, capsid, and matrix protein of the HIV-1 virus were labeled with a Ru(II) complex ([Ru(phen)2(dppz)](2+)), the TC-FlAsH/ReAsH system, and EGFP/ECFP, respectively. Using the multicolored virus and single-particle imaging, we were able to track the sequential disassembly process of single HIV-1 virus particles in live host cells. Approximately 0.1% of viral particles were observed to undergo a sequential disassembly process at 60-120 min post infection. The timing and efficiency of the disassembly were influenced by the cellular factor CypA and reverse transcription. The findings facilitate a better understanding of the processes governing the HIV-1 lifecycle. The multicolor labeling protocol developed in this study may find many applications involving virus-host-cell interactions.

Intranasal Nanovaccine Confers Homo- and Hetero-Subtypic Influenza Protection

Cross-protective and non-invasively administered vaccines are attractive and highly desired for the control of influenza. Self-assembling nanotechnology provides an opportunity for the development of vaccines with superior performance. In this study, an intranasal nanovaccine is developed targeting the conserved ectodomain of influenza matrix protein 2(M2e). 3-sequential repeats of M2e (3M2e) is presented on the self-assembling recombinant human heavy chain ferritin (rHF) cage to form the 3M2e-rHF nanoparticle. Intranasal vaccination with 3M2e-rHF nanoparticles in the absence of an adjuvant induces robust immune responses, including high titers of sera M2e-specific IgG antibodies, T-cell immune responses, and mucosal secretory-IgA antibodies in mice. The 3M2e-rHF nanoparticles also confer complete protection against a lethal infection of homo-subtypic H1N1 and hetero-subtypic H9N2 virus. An analysis of the mechanism of protection underlying the intranasal immunization with the 3M2e-rHF nanoparticle indicates that M2e-specific mucosal secretory-IgA and T-cell immune responses may play critical roles in the prevention of infection. The results suggest that the 3M2e-rHF nanoparticle is a promising, needle-free, intranasally administered, cross-protective influenza vaccine. The use of self-assembling nanovaccines could be an ideal strategy for developing vaccines with characteristics such as high immunogenicity, cross-protection, and convenient administration, as well as being economical and suitable for large-scale production.

Live cell imaging of single genomic loci with quantum dot-labeled TALEs

Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labelling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different colour quantum dots (QDs). These QD-labelled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different coloured QD-labelled TALEs, we determine and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.

Isolation and characterization of a Far-Eastern strain of tick-borne encephalitis virus in China.

Tick-borne encephalitis virus (TBEV) is a leading cause of human neurological infection in many parts of Europe and Asia. Although several TBEV isolates have been reported, current understanding of the biological characteristics of a Chinese strain is limited. In this study, a Far-Eastern strain of TBEV designated WH2012 was isolated in northern China. Its genome has been sequenced and found to be closely related to other Chinese TBEV isolates. Human cell lines of neural origin exposed to WH2012 showed cytopathic effects and WH2012 replicated most efficiently in human neuroblastoma cells SK-N-SH. In addition, WH2012 possessed a pathogenic potential in the mouse model, characterized by inducing a complete paralysis in the hindlimbs with a fatal outcome. We herein describe the first data regarding biological properties of TBEV from China. This study may help future research on pathogenic mechanisms of the neurological disease induced by TBEV infection in China.