Leike Zhang

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

ABSTRACT The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently.

The metabolic responses to hepatitis B virus infection shed new light on pathogenesis and targets for treatment

Chronic infection caused by the hepatitis B virus (HBV), is strongly associated with hepatitis, fatty liver and hepatocellular carcinoma. To investigate the underlying mechanisms, we characterize the metabolic features of host cells infected with the virus using systems biological approach. The results show that HBV replication induces systematic metabolic alterations in host cells. HBV infection up-regulates the biosynthesis of hexosamine and phosphatidylcholine by activating glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1) and choline kinase alpha (CHKA) respectively, which were reported for the first time for HBV infection. Importantly suppressing hexosamine biosynthesis and phosphatidylcholine biosynthesis can inhibit HBV replication and expression. In addition, HBV induces oxidative stress and stimulates central carbon metabolism and nucleotide synthesis. Our results also indicate that HBV associated hepatocellular carcinoma could be attributed to GFAT1 activated hexosamine biosynthesis and CHKA activated phosphatidylcholine biosynthesis. This study provides further insights into the pathogenesis of HBV-induced diseases, and sheds new light on drug target for treating HBV infection.

Identification of host proteins involved in Japanese encephalitis virus infection by quantitative proteomics analysis.

Japanese encephalitis virus (JEV) enters host cells via receptor-mediated endocytosis and replicates in the cytoplasm of infected cells. To study virus-host cell interactions, we performed a SILAC-based quantitative proteomics study of JEV-infected HeLa cells using a subcellular fractionation strategy. We identified 158 host proteins as differentially regulated by JEV (defined as exhibiting a greater than 1.5-fold change in protein abundance upon JEV infection). The mass spectrometry quantitation data for selected proteins were validated by Western blot and immunofluorescence confocal microscopy. Bioinformatics analyses were used to generate JEV-regulated host response networks consisting of regulated proteins, which included 35 proteins that were newly added based on the results of this study. The JEV infection-induced host response was found to be coordinated primarily through the immune response process, the ubiquitin-proteasome system (UPS), the intracellular membrane system, and lipid metabolism-related proteins. Protein functional studies of selected host proteins using RNA interference-based techniques were carried out in HeLa cells infected with an attenuated or a highly virulent strain of JEV. We demonstrated that the knockdown of interferon-induced transmembrane protein 3 (IFITM3), Ran-binding protein 2 (RANBP2), sterile alpha motif domain-containing protein 9 (SAMD9) and vesicle-associated membrane protein 8 (VAMP8) significantly increased JEV replication. The results presented here not only promote a better understanding of the host response to JEV infection but also highlight multiple potential targets for the development of antiviral agents.

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.

Activation of the RLR/MAVS Signaling Pathway by the L Protein of Mopeia Virus

ABSTRACT The family Arenaviridae includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of type I interferon (IFN-I). However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the L polymerase (Lp) of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and for the elucidation of arenavirus pathogenesis. IMPORTANCE Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of the RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP, and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or reduced. Our study provides a possible explanation for how IFN-I can become activated during arenavirus infection and may help us gain insights into the interactions that form between different arenavirus components and the innate immune system.

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.

Analysis of EV71 infection progression using triple‐SILAC‐based proteomics approach

Enterovirus 71 (EV71), a member of Picornaviridae, causes severe neurological and systemic illness in children. To better understand the virus–host cell interactions, we performed a triple‐SILAC‐based quantitative proteomics study monitoring host cell proteome changes after EV71 infection. Based on the quantitative data for more than 4100 proteins, ∼17% of the proteins were found as significantly changed (p<0.01) at either 8 or 20 hours post infection. Five biological processes and seven protein classes showed significant differences. Functional screening of nine regulated proteins discovered the regulatory role of CHCH2, a mitochondrial protein known as a transcriptional activator for cytochrome c oxidase, in EV71 replication. Further studies showed that CHCH2 served as a negative regulator of innate immune responses. All MS data have been deposited in the ProteomeXchange with identifier PXD002483 (http://proteomecentral.proteomexchange.org/dataset/PXD002483).