Xiang Mao

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication.

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.

Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions.

Abstract Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5′ and 3′ un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections.

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

Abstract Japanese encephalitis virus (JEV), one of the causes for epidemic encephalitis, belongs to the family of Flaviviridae. In this study, we demonstrated that cellular DEAD-box RNA helicase DDX5 plays an important role in JEV replication. The knockdown of DDX5 was able to decrease JEV replication, and overexpression of DDX5 mutants lacking the helicase activity also reduced JEV replication, suggesting the helicase activity is essential for JEV replication. DDX5 knockdown did not affect virus assembly and release. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX5 could interact with JEV core protein, non-structural protein 3 (NS3) and 5 (NS5-MTase and NS5-RdRp domains). Meanwhile, we also confirmed that DDX5 interacts with these viral proteins during JEV infection. Confocal microscopy analysis showed that endogenous DDX5 is recruited to the cytoplasm and colocalizes with these viral proteins and viral RNA. RNA-pulldown experiment showed that DDX5 only binds to the JEV 3′ untranslated region (UTR). Finally, we confirmed the role of DDX5 in JEV RNA replication using JEV-replicon system. In conclusion, we identified DDX5 as a positive regulator for JEV replication.

Inhibition of Japanese encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase ribosomal-inactivating protein isolated from Phytolacca americana. The antiviral activity of PAP has been described in several viruses. This study was to investigate the antiviral activity of PAP against Japanese encephalitis virus (JEV) infection in vitro and in vivo. Antiviral activity of PAP against JEV infection was evaluated in vitro using plaque forming assay, qRT-PCR and Western blot analysis. In vitro results showed that PAP inhibited replication of JEV in a dose-dependent manner with 50% inhibitory concentration (IC(50)) of 300 ng/ml (23.1 nM). Depurination assay suggested that the antiviral activity of PAP against JEV infection might be partially due to depurination of JEV genomic RNA. In vivo studies showed that PAP (1.0mg/kg) administered intraperitoneally decreased infection in mice challenged with a lethal dose of JEV, presenting a survival of 87.5% or 85.7% when administered pre-infection or post-infection. Collectively, our studies demonstrated that PAP possesses antiviral activity against JEV infection in vitro and in vivo, providing evidences for further development of PAP as an antiviral agent against JEV infection.

Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII

Japanese encephalitis virus (JEV) infection is a major cause of acute viral encephalitis both in humans and animals. The domain III of virus envelope protein (EDIII) plays important roles in interacting with host cell receptors to facilitate virus entry. In this study, recombinant JEV EDIII was expressed and purified. The protein showed the ability to inhibit JEV infection in BHK-21 cells with 50% inhibition at a concentration of 25μg/ml. Based on NMR structure of JEV EDIII, we chose several loop peptides that were reported to be related to receptor binding to test their possible inhibitory activities on virus infection. Our in vitro experiments demonstrated that one of the loop peptides (loop3) can prevent JEV infection with 50% inhibition at concentration of 10μM by interfering in virus attachment to the cells. Our in vivo experiments on mice showed the loop3 was the most protective peptide when administered before virus challenge. Therefore, the loop3 peptide may be served as basis for the development of novel antiviral agents against Japanese encephalitis virus or other flaviviruses infection.

Isolation and potential immunological characterization of TPSGLVY, a novel bursal septpeptide isolated from the bursa of Fabricius

The bursa of Fabricius is central immune organ unique to birds, and the extract is immunocompetent in stimulating B cell differentiation and enhancing antibody production. However, except for bursin, the active peptides from the bursa of Fabricius are little reported. In the paper, a novel bursal septpeptide (BSP-II) with the amino acids sequence of TPSGLVY was identified and similar to the MGC53864 protein of Gallus gallus. We investigated the effects of BSP-II on the immune response in terms of the antibodies titers (IgG1 and IgG2alpha), the levels of interferon-gamma and interleukin-4 cytokines, spleen cell lymphocyte proliferation, and the T-lymphocyte subtype composition. It was noteworthy that BSP-II potentiates the Th1 and Th2-type immune responses in dose-dependent manner. BSP-II had specific enhancing effects on the hybridoma SP2/0 cell proliferation at two different serum concentrations (20% and 5%), but had no connection with the dose of BSP-II. The antibody secreting level of hybridoma SP2/0 cells rose in 5% and 20% serum when the concentrations of BSP-II increased. Also, BSP-II had effect on the viabilities of tumor cells (Hela and SP2/0). All the results indicated that BSP-II was able to significantly induce various immune responses and involved in the cell viability of different tumor cell lines. Our observations implied that BSP-II might be a novel biological active factor from the bursa of Fabricius with immunomodulatory activities.

Porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor

It is well known that many viruses use heparan sulfate as the initial attachment factor. In the present study, we determined whether porcine epidemic diarrhea virus (PEDV), an emerging veterinary virus, infects Vero cells by attaching to heparan sulfate. Western blot analysis, real-time PCR, and plaque formation assay revealed that PEDV infection was inhibited when the virus was pretreated with heparin (an analogue of heparan sulfate). There was no inhibitory effect when the cells were pre-incubated with heparin. We next demonstrated that enzymatic removal of the highly sulfated domain of heparan sulfate by heparinase I treatment inhibited PEDV infection. We also confirmed that sodium chlorate, which interferes with heparan sulfate biosynthesis, also inhibited PEDV infection. Furthermore, we examined the effect of two heparin derivatives with different types of sulfation on PEDV infection. The data suggested de-N-sulfated heparin, but not N-acetyl-de-O-sulfated heparin, inhibits PEDV infection. In summary, our studies revealed that heparan sulfate acts as the attachment factor of PEDV in Vero cells.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

Structure and Function Analysis of Nucleocapsid Protein of Tomato Spotted Wilt Virus Interacting with RNA Using Homology Modeling

Background: The crystal structure of viral proteins is not available for many plant viruses including tomato spotted wilt virus (TSWV). Results: By homology modeling, we mapped the RNA binding sites and discovered a protective feature of TSWV nucleocapsid (N). Conclusion: Homology modeling provided a basis for functional analysis of TSWV N interacting with RNA. Significance: This approach might be applicable for other plant viruses. The nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) plays key roles in assembling genomic RNA into ribonucleoprotein (RNP), which serves as a template for both viral gene transcription and genome replication. However, little is known about the molecular mechanism of how TSWV N interacts with genomic RNA. In this study, we demonstrated that TSWV N protein forms a range of higher ordered oligomers. Analysis of the RNA binding behavior of N protein revealed that no specific oligomer binds to RNA preferentially, instead each type of N oligomer is able to bind RNA. To better characterize the structure and function of N protein interacting with RNA, we constructed homology models of TSWV N and N-RNA complexes. Based on these homology models, we demonstrated that the positively charged and polar amino acids in its predicted surface cleft of TSWV N are critical for RNA binding. Moreover, by N-RNA homology modeling, we found that the RNA component is deeply embedded in the predicted protein cleft; consistently, TSWV N-RNA complexes are relatively resistant to digestion by RNase. Collectively, using homology modeling, we determined the RNA binding sites on N and found a new protective feature for N protein. Our findings also provide novel insights into the molecular details of the interaction of TSWV N with RNA components.

LiCl inhibits PRRSV infection by enhancing Wnt/β-catenin pathway and suppressing inflammatory responses.

Lithium chloride (LiCl) has been used as a mood stabilizer in the manic depressive disorders treatment. Recent studies show that LiCl is also a potent inhibitor for some DNA and RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen in modern pig industry. In this study, we assessed the inhibitory effect of LiCl on PRRSV infection using plaque-formation assay, Q-PCR and Western blot analysis. Our results showed that LiCl could inhibit PRRSV infection in MARC-145 and PAM-CD163 cells. Previous reports have shown that LiCl could induce the Wnt pathway in the absence of Wnt ligands. In our studies, we demonstrated that LiCl activates the Wnt pathway in PRRSV infected cells. Additionally, the knockdown of β-catenin or the Wnt/β-catenin pathway inhibitor PNU74654 was able to reverse the antiviral effect of LiCl, which suggested that the inhibitory effect of LiCl against PRRSV replication might be associated with the activation of the Wnt/β-catenin pathway. We also found that lower viral replication after LiCl treatment was associated with the reduced mRNA levels of pro-inflammatory IL-8, IL-6, IL-1 β, tumor necrosis factor α and decreased NF-κB nuclear translocation. Collectively, our data demonstrated that LiCl inhibited PRRSV infection by enhancing Wnt/β-catenin pathway and suppressing pro-inflammatory responses.