Ji-Kai Wen

Imaging Viral Behavior in Mammalian Cells with Self-Assembled Capsid-Quantum-Dot Hybrid Particles

Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long-term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD-containing virus-like particles (VLPs) of simian virus 40 (SV40), termed SVLP-QDs, are constructed by in vitro self-assembly of the major capsid protein of SV40. SVLP-QDs show homogeneity in size ( approximately 24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP-QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.

Phage display mediated immuno-PCR

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10 000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

Visualizing the dynamic behavior of poliovirus plus-strand RNA in living host cells

Dynamic analysis of viral nucleic acids in host cells is important for understanding virus–host interaction. By labeling endogenous RNA with molecular beacon, we have realized the direct visualization of viral nucleic acids in living host cells and have studied the dynamic behavior of poliovirus plus-strand RNA. Poliovirus plus-strand RNA was observed to display different distribution patterns in living Vero cells at different post-infection time points. Real-time imaging suggested that the translocation of poliovirus plus-strand RNA is a characteristic rearrangement process requiring intact microtubule network of host cells. Confocal-FRAP measurements showed that 49.4 ± 3.2% of the poliovirus plus-strand RNA molecules diffused freely (with a D-value of 9.6 ± 1.6 × 10−10 cm2/s) within their distribution region, while the remaining (50.5 ± 2.9%) were almost immobile and moved very slowly only with change of the RNA distribution region. Under the electron microscope, it was found that virus-induced membrane rearrangement is microtubule-associated in poliovirus-infected Vero cells. These results reveal an entrapment and diffusion mechanism for the movement of poliovirus plus-strand RNA in living mammalian cells, and demonstrate that the mechanism is mainly associated with microtubules and virus-induced membrane structures.

A visual DNA chip for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1.

For the simultaneously visual detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-1), a qualitative DNA chip method, combining multiplex and nested polymerase chain reaction (PCR) with arrayed anchored primer PCR and a biotin-avidin alkaline phosphatase (Av-AP) indicator system, was developed. After pretreatment of infected blood samples and reverse transcription of the RNA virus genome, PCR was performed in a single tube by using the outer primer pairs. Second round nested multiplex PCR was performed on the DNA chip, on which the primers array had already been prepared. During the arrayed anchored multiplex PCR, 5[N-(N-biotinylaminocaproyl)-epsilon-3-aminoallyl]-2-deoxy-uridine-5-triphosphate (biotin-11-dUTP) was incorporated into the extended DNA chains in order to bind avidin alkaline phosphatase via avidin and biotin. To produce purple precipitates on the chips, the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was used in conjunction with the enhancer, nitro blue tetrazolium (NBT). Blood samples containing the three viruses were tested using this DNA chip and about 1 pg of specific viral DNA fragments were detected on the chip wells after nested PCR.

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9+/-0.4% (n=36), 4.5+/-0.4% (n=39), and 8.3+/-0.6% (n=44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7+/-1.1% (n=39) and 9.0+/-0.9% (n=37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.

Construction and characterization of an anti-prion scFv fusion protein pair for detection of prion protein on antibody chip

Abstract A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrPc). After five rounds of panning against recombinant bovine prion protein (rb‐PrPc) and ELISA test, two positive clones with high affinity to rb‐PrPc, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrPc were 3.24×10−8 M, 8.82×10−8M, and 8.10×10−9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrPc with detection limit of 1 pg/ml.