Shaobo Wang

Japanese Encephalitis Virus Activates Autophagy as a Viral Immune Evasion Strategy

In addition to manipulating cellular homeostasis and survivability, autophagy also plays a crucial role in numerous viral infections. In this study, we discover that Japanese encephalitis virus (JEV) infection results in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II) protein and GFP-LC3 puncta in vitro and an increase in autophagosomes/autolysosomes in vivo. The fusion between autophagosomes and lysosomes is essential for virus replication. Knockdown of autophagy-related genes reduced JEV replication in vitro, as indicated by viral RNA and protein levels. We also note that JEV infection in autophagy-impaired cells displayed active caspases cleavage and cell death. Moreover, we find that JEV induces higher type I interferon (IFN) activation in cells deficient in autophagy-related genes as the cells exhibited increased phosphorylation and dimerization of interferon regulatory factor 3 (IRF3) and mitochondrial antiviral signaling protein (MAVS) aggregation. Finally, we find that autophagy is indispensable for efficient JEV replication even in an IFN-defective background. Overall, our studies provide the first description of the mechanism of the autophagic innate immune signaling pathway during JEV infection.

Peptide inhibitor of Japanese encephalitis virus infection targeting envelope protein domain III.

Japanese encephalitis virus (JEV) is a major cause of acute viral encephalitis in both humans and animals. Domain III of the virus envelope glycoprotein (E DIII) plays an important role in the interaction of viral particles with host cell receptors to facilitate viral entry. Intervention of the interaction between E DIII and its cognate host cell receptor would provide an important avenue for inhibiting JEV infection. A phage display peptide library was therefore panned against E DIII, which resulted in the identification of several peptides. One peptide, named P3, inhibited JEV infection of BHK-21 cells with an IC₅₀ of ∼1 μM and an IC₉₀ at ∼100 μM. Further characterization revealed that P3 bound to E DIII with a K(d) of 6.06 × 10⁻⁶ M and inhibited JEV infection by interfering with viral attachment to cells. Based on in silico prediction by ZDOCK, P3 was found to interact with E DIII via a hydrophobic pocket, which was confirmed by the binding assay of P3 to the V357A mutant. P3 was hypothesized to bind to E DIII by interacting with the sties adjacent to the BC and DE loops, which might interfere with the binding of JEV to cellular receptors, thus impeding viral infection. This newly isolated peptide may represent a new therapeutic candidate for treatment of JEV.

Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

ABSTRACT Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I0, ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity. IMPORTANCE Recently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

Antiviral activity of peptide inhibitors derived from the protein E stem against Japanese encephalitis and Zika viruses

ABSTRACT Japanese encephalitis virus (JEV) and Zika virus (ZIKV) are mosquito‐borne viruses of the Flavivirus genus that cause viral encephalitis and congenital microcephaly, respectively, in humans, and thus present a risk to global public health. The envelope glycoprotein (E protein) of flaviviruses is a class II viral fusion protein that mediates host cell entry through a series of conformational changes, including association between the stem region and domain II leading to virion‐target cell membrane fusion. In this study, peptides derived from the JEV E protein stem were investigated for their ability to block JEV and ZIKV infection. Peptides from stem helix 2 inhibit JEV infection with the 50% inhibitory concentration (IC50) in the nanomolar range. One of these peptides (P5) protected mice against JEV‐induced lethality by decreasing viral load, while abrogating histopathological changes associated with JEV infection. We also found that P5 blocked ZIKV infection with IC50 at the micromolar level. Moreover, P5 was proved to reduce the histopathological damages in brain and testes resulting from ZIKV infection in type I and II interferon receptor‐deficient (AG6) mice. These findings provide a basis for the development of peptide‐based drugs against JEV and ZIKV. HighlightsPeptides derived from the JEV E protein stem region prevented JEV and ZIKV infection.Peptides from stem helix 2 inhibit JEV and ZIKV infection with IC50 in the nanomolar and micromolar range, respectively.P5 protected mice against JEV‐induced lethality by decreasing viral load.P5 blocked ZIKV infection in vivo by decreasing viral load and abrogating histopathological changes both in brain and testes.

Subcellular quantitative proteomic analysis reveals host proteins involved in human cytomegalovirus infection.

Viral replication requires host cell macromolecules and energy, although host cells can alter their protein expression to restrict viral replication. To study the host cell response to human cytomegalovirus (HCMV) infection, a stable isotope labeling by amino acids in cell culture (SILAC)-based subcellular quantitative proteomic study of HCMV-infected human embryo lung fibroblast (HEL) cells was performed, and a total of 247 host proteins were identified as differentially regulated by HCMV. Western blotting and immunofluorescence confocal microscopy were performed to validate the data sets. Gene Ontology analysis indicated that cellular processes involving the metabolism, localization and immune system were regulated as a result of HCMV infection. Functional analysis of selected regulated proteins revealed that knockdown of HNRPD, PHB2 and UB2V2 can increase HCMV replication, while knockdown of A4 and KSRP resulted in decreased HCMV replication. Our study may improve our understanding of the dynamic interactions between HCMV and its host and provide multiple potential targets for anti-HCMV agent research.

Quantitative proteomic analysis of mosquito C6/36 cells reveals host proteins involved in Zika virus infection

ABSTRACT Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae. During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo. Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo. Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

Methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant of the human prion protein

The D178N mutation of the prion protein (PrP) results in the hereditary prion disease fatal familial insomnia (FFI). Little is known regarding the effects of methionine oxidation on the pathogenesis of D178N-associated FFI. In the present study, we found that the D178N variant was more susceptible to oxidation than wild-type PrP, as indicated by reverse-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) analysis. Circular dichroism (CD), differential scanning calorimetry (DSC), thioflavin T (ThT) binding assay studies demonstrated that methionine oxidation decreased the structural stability of the D178N variant, and the oxidized D178N variant exhibited a greater propensity to form β-sheet-rich oligomers and aggregates. Moreover, these aggregates of oxidized D178N PrP were more resistant to proteinase K (PK) digestion. Additionally, using fluorescence confocal microscopy, we detected a high degree of aggregation in D178N-transfected Neuro-2a (N2a) cells after treatment with hydrogen peroxide (H2O2). Furthermore, the oxidation and consequent aggregation of the D178N variant induced greater apoptosis of N2a cells, as monitored using flow cytometry. Collectively, these observations suggest that methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant, possibly providing direct evidence to link the pathogenesis of D178N-associated FFI with methionine oxidation.

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection

ABSTRACT Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca2+ channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.

The ubiquitin-proteasome system is essential for the productive entry of Japanese encephalitis virus.

The host-virus interaction during the cellular entry of Japanese encephalitis virus (JEV) is poorly characterized. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. Here, we showed that the proteasome inhibitors, MG132 and lactacystin, impaired the productive entry of JEV by effectively interfering with viral intracellular trafficking at the stage between crossing cell membrane and the initial translation of the viral genome after uncoating. Using confocal microscopy, it was demonstrated that a proportion of the internalized virions were misdirected to lysosomes following treatment with MG132, resulting in non-productive entry. In addition, using specific siRNAs targeting ubiquitin, we verified that protein ubiquitination was involved in the entry of JEV. Overall, our study demonstrated the UPS is essential for the productive entry of JEV and might represent a potential antiviral target for JEV infection.

Structure-function relationship of the mammarenavirus envelope glycoprotein

Mammarenaviruses, including lethal pathogens such as Lassa virus and Junín virus, can cause severe hemorrhagic fever in humans. Entry is a key step for virus infection, which starts with binding of the envelope glycoprotein (GP) to receptors on target cells and subsequent fusion of the virus with target cell membranes. The GP precursor is synthesized as a polypeptide, and maturation occurs by two cleavage events, yielding a tripartite GP complex (GPC) formed by a stable signal peptide (SSP), GP1 and GP2. The unique retained SSP interacts with GP2 and plays essential roles in virion maturation and infectivity. GP1 is responsible for binding to the cell receptor, and GP2 is a class I fusion protein. The native structure of the tripartite GPC is unknown. GPC is critical for the receptor binding, membrane fusion and neutralization antibody recognition. Elucidating the molecular mechanisms underlining the structure–function relationship of the three subunits is the key for understanding their function and can facilitate novel avenues for combating virus infections. This review summarizes the basic aspects and recent research of the structure–function relationship of the three subunits. We discuss the structural basis of the receptor-binding domain in GP1, the interaction between SSP and GP2 and its role in virion maturation and membrane fusion, as well as the mechanism by which glycosylation stabilizes the GPC structure and facilitates immune evasion. Understanding the molecular mechanisms involved in these aspects will contribute to the development of novel vaccines and treatment strategies against mammarenaviruses infection.