Justin Jang Hann Chu

Photodynamic inactivation of viruses using upconversion nanoparticles

Photodynamic therapy (PDT) is a promising treatment modality that utilizes light of an appropriate wavelength to excite photosensitive materials called photosensitizers, which upon excitation, generate reactive oxygen species (ROS) that are cytocidal and virucidal. However, problems such as hydrophobicity of photosensitizers and limited tissue penetration ability of the current light sources impeded its promotion as a mainstay in medical technology. Here, by using near-infrared (NIR)-to-visible upconversion nanoparticles (UCNs), we demonstrate UCN-based photodynamic inactivation as a potential antiviral strategy. These UCNs are nanotransducers which not only act as carriers of photosensitizers but also active participants in PDT by transducing NIR radiation to visible emissions appropriate for excitation of the attached photosensitizers. The UCNs effectively reduced the infectious virus titers in vitro with no clear pathogenicity in murine model and increased target specificity to virus-infected cells. Hence, this is a promising antiviral approach with feasible applications in the treatments of virus-associated infections, lesions and cancers.

Quantifying the Specific Binding between West Nile Virus Envelope Domain III Protein and the Cellular Receptor αVβ3 Integrin

A previous study has illustrated that the αVβ3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to αVβ3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to αVβ3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, αVβ3 integrin. The single protein pair-interacting force measured was in the range of 45 ± 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with αVβ5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Developments towards antiviral therapies against enterovirus 71.

Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. Recurring outbreaks of EV71 have been reported in several countries. The current lack of approved anti-EV71 therapy has prompted intense research into antiviral development. Several strategies – ranging from target-based chemical design to compound library screenings – have been employed, while others revisited compound series generated from antiviral developments against poliovirus and human rhinoviruses. These efforts have given rise to a diversity of antiviral candidates that include small molecules and non-conventional nucleic-acid-based strategies. This review aims to highlight candidates with potential for further clinical development based on their putative modes of action.

The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 μm chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.

Inhibition of Chikungunya Virus Replication by Harringtonine, a Novel Antiviral That Suppresses Viral Protein Expression

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting ≥70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC50] = 0.24 μM) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.

Exposure to Titanium Dioxide Nanoparticles Induces Autophagy in Primary Human Keratinocytes

Understanding the mechanisms of cell-nanomaterial interactions is vital in harnessing the potential of using nanomaterials in biomedical applications. By immuno-labeling of LC3 and TEM analysis, it is found that titanium dioxide nanoparticles are internalized by human keratinocytes and induce autophagy. Autophagy appears to play a cytoprotective role in response to toxicity influence exerted by the nanoparticles.

Chikungunya virus: an update on antiviral development and challenges

Chikungunya virus (CHIKV) has re-emerged as a significant public health threat since the 2005 chikungunya fever epidemic in La Réunion. Driven by the medical importance of this virus, as well as the lack of approved antivirals, research into the field of CHIKV antivirals has recently intensified. Potential therapeutics that have been reported to show anti-CHIKV activity in vitro range from known broad-spectrum antivirals like chloroquine to novel strategies involving RNA silencing technology. Although most of the earlier efforts focused on compounds that target host components, some recent studies have reported viral targets such as nonstructural proteins. This article examines the reported in vitro and in vivo efficacies, as well as the therapeutic potential of these antiviral compounds.

The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery

The polypyrimidine tract-binding protein (PTB) functions primarily as an IRES trans-acting factor in the propagation of hepatitis C virus and picornaviruses. PTB interacts with secondary structures within the 3′- and 5′-untranslated regions of these viral genomes to mediate efficient IRES-mediated viral translation. PTB has also been reported to bind to the untranslated region of the single-stranded RNA dengue virus (DENV), suggesting a similar function for PTB in flaviviruses. Indeed small interfering RNA-mediated PTB knockdown inhibited the production of infectious DENV, and this inhibition was specific to PTB knockdown and not due to a nonspecific anti-viral state. In fact, PTB depletion did not inhibit the production infectious yellow fever virus, another flavivirus. Nevertheless, whereas PTB knockdown led to a significant decrease in the accumulation of DENV viral RNAs, it did not impair translation. Moreover, PTB was shown to interact with the DENV nonstructural 4A protein, a known component of the viral replication complex, and with the DENV genome during infection. These data suggest that PTB interacts with the replication complex of DENV and is acting at the level of viral RNA replication.

Cellular Vimentin Regulates Construction of Dengue Virus Replication Complexes through Interaction with NS4A Protein

ABSTRACT Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.

Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

High mobility group box 1 (HMGB1) protein is released from cells as a pro-inflammatory cytokine in response to an injury or infection. During dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a number of pro-inflammatory cytokines are released, contributing to disease pathogenesis. In this study, the release of HMGB1 from human myelogenous leukemia cell line K562 and primary peripheral blood monocytes (PBM) cells was examined during dengue virus (DV)-infection. HMGB1 was shown to translocate from cell nuclei to the cytoplasm in both K562- and PBM-infected cells. The translocation of HMGB1 from the nucleus to the cytoplasm was shown to be mediated by the host cell p300/CBP-associated factor (PCAF) acetylase complex in K562 cells. In addition, DV capsid protein was observed to be the putative viral protein in actuating HMGB1 migration from the nucleus to cytoplasm through the involvement of PCAF acetylase. HMGB1 was released from DV-infected K562 cells into the extracellular milieu in a multiplicity of infection (M.O.I.)-independent manner and its release can be inhibited by the addition of 1–5 mM of ethyl pyruvate (EP) in a dose-dependent manner. Application of DV-infected K562 cell culture supernatants to primary endothelial cells induced vascular permeability. In contrast, supernatants from DV-infected K562 cells treated with EP or HMGB1 neutralizing antibody were observed to maintain the structural integrity of the vascular barrier.