Studies On Cell Entry Of Japanese Encephalitis Virus Into Human Neuronal Cells

Background and Aim:Japanese encephalitis virus(JEV) is an important human pathogen, causing Japanese encephalitis(JE) in Asia, western Pacific countries, and northern Australia. It is estimated that approximately 35,000-50,000 JE cases have occurred annually among countries where JE is epidemic. The fatality rate in JE cases ranges from 20% to 30%, and 30%-50% of survivors are left with severe neurological sequelae. Thus, JEV still has the potential to become a worldwide public health threat. Understanding the mechanisms of the early events of viral infection and identifying new antiviral targets will provide more approaches for JE prevention and treatment.Initiation of viral infection requires entry of the virus into the host cell,which is an early event and critical to the replication of virus. JEV E is the major structural protein exposed on the surface of the particle and is suggested to be engaged in viral attachment, penetration, and membrane fusion. JEV infection occurs by binding of viral E glycoprotein with a specific receptor(s) on the cell membrane, which subsequently leads to internalization of the virus into cells. Although it has been widely accepted that JEV enters permissive cells via receptor-mediated endocytosis, the identity of the cellular receptor interacting with E glycoprotein is, at present, controversial.Numerous animal viruses exploit the cellular endocytic mechanisms for entry into target cells. Since JEV is a neurotropic virus and infection of JEV induced extensive inflammation in the central nervous system is the main cause of viral encephalitis, the discovery of cellular molecules and pathways that mediate JEV entry into neuronal cells is important. Previous reports showed that JEV was endocytosed into Vero, C6/36 cells, and mouse neural stem cells by a clathrin-dependent pathway. Conversely, it was recently reported that JEV infects neuronal cells through a clathrin-independent endocytic mechanism. We have also shown that JEV enters rat neuroblastoma cells via a caveolae-mediated endocytosis pathway, which may be the specific endocytic manner for JEV infection of neuronal cells. Although these studies have identified the entry processes of JEV into neuronal cells, the specific cellular factors and pathways utilized in this process have not been fully characterized.Here, we assessed an array of si RNA libraries targeting 140 cellular membrane-trafficking genes and systematically evaluated their impact on the infectious entry of JEV into human SK-N-SH neuroblastoma cells. The RNAi analysis, in combination with specific inhibitors, dominant-negative mutants, and microscopy were used to examine the key cellular molecules involved in JEV entry.Part I: Identification of membrane-trafficking genes and endocytosis pathways modulate JEV entry.Materials & methods: To identify cellular factors involved in JEV entry to SK-N-SH cells, a targeted si RNA silencing screen using the human endocytic/membrane trafficking library from Dharmacon was performed. We first identified si RNAs that inhibited the JEV wild-type strain SA14 infection as a primary evaluation screen. We then re-tested genes having an inhibitory effect on JEV infection in the primary screen using a pseudotype JEV(JEVpv) system. Then, specific inhibitors, dominant-negative mutants, and microscopy were used to examine endocytosis pathways for JEV entry into SK-N-SH cells.Results: A total of 23 human genes that have an inhibitory effect on JEV infection were obtained in our primary screen. A total of 15 host genes were identified as key molecules for JEV entry The genes were classified into many different protein classes. Silencing of caveolin-1, the major defining marker of caveolae and the crucial driver of caveolae formation, led to a decrease in viral entry. The si RNAs targeting kinases(PIK3CG, PIK3C2G) involved in the signal transduction processes of viral entry were also found to reduce JEV entry. Knockdown of human genes essential in regulating actin polymerization(CFL1, PAK1, RAC1, RHOA and ROCK2) displayed inhibitory effects on JEV entry. Silencing of genes involved in vesicle and endosomal transport such as COPA, RAB5 A, RAB5 C and VAPA also led to a decrease in viral entry. Furthermore, si RNA knockdown of dynamin-2, a large GTPase which acts by mediating the release of newly formed endocytic vesicles from the plasma membrane, also exhibited inhibitory effect on JEV entry. JEV entry into SK-N-SH cells depends on caveolin-1 and dynamin-2, but independent of components associated with clathrin-mediated endocytosis or macropinocytosis. JEV entry induced an increase in caveolin-1 phosphorylation. In JEV-infected cells, subcellular localization analysis showed that a large proportion of incoming JEV virions colocalize with caveolin-1. Intracellular virus transport is dependent on Rab5-positive early endosomes but is independent of Rab7 late endosomes.Conclusion: JEV enters human neuronal cells through a caveolin-1-mediated and dynamin-2-dependent endocytic pathway. After internalization, JEV required transport to Rab5-positive endosomes.Part II: Mechanisms of actin reorganization in JEV entry of human neuronal cells.Materials & methods: Si RNA, specific inhibitors and dominant-negative mutants for Rho GTPases and their downstream regulators were used to examine the role of actin reorganization during JEV entry into SK-N-SH cells.Results: We found that JEV entry into neuronal SK-N-SH cells induced dynamic rearrangements of the actin cytoskeleton at the early stages of infection. JEV-induced actin rearrangements were required for caveolin-1-mediated JEV endocytosis. Rho GTPases family members Rho A and Rac1, but not Cdc42, play a critical role in virus entry. Rho A and Rac1 induced early actin rearrangement during JEV entry through the modulation of cofilin activity. Although Rho A and Rac1 are both required for viral entry by modulating steps of post-binding events, they exert their effects at different times in the viral internalization process, virus-induced Rho A activation occurs earlier than Rac1. In addition, Rho A and Rac1 exert different effects in caveolin-1-mediated JEV entry. Rho A activation is required for JEV-induced caveolin-1 phosphorylation, while Rac1 is activated by phosphorylated caveolin-1 in JEV entry processes. Moreover, Nocodazole treatment shows that microtubules play an important role in the JEV entry processes after the initial viral internalization.Conclusion: JEV enters human neuronal cells depends on a two-step regulation of actin cytoskeleton remodeling triggered by Rho A and Rac1: Rho A activation promoted the phosphorylation and aggregation of caveolin-1 on the cell membrane, and then Rac1 activation facilitated caveolin-associated viral internalization.Part III: Mechanisms of epidermal growth factor receptor in JEV entry of human neuronal cells.Materials & methods: Si RNA and specific inhibitors targeting EGFR and its downstream kinase PI3 K were used to investigate the role of EGFR and downstream signaling pathway in JEV entry. Immunoprecipitation and microscopy were also used to examine the role of lipid rafts for efficient JEV entry.Results: Genistein treatment shows that tyrosine kinases are required for caveolin-1-mediated JEV entry. JEV induced EGFR phosphorylation upon JEV infection. Pre-incubation of cells with EGF induced EGFR phosphorylation and enhanced JEV internalization. Depletion of EGFR by specific si RNA or inhibition of the EGFR kinase by an EGFR specific inhibitor erlotinib both reduced JEVpv entry and JEV internalization, indicating EGFR is required for JEV entry. kinetics entry assay showed that EGFR acts as a membrane mediator for JEV entry event initiation. PI3K/Akt, the direct downstream kinase of EGFR, was involved in JEV entry. Either EGFR or PI3 K knockdown inhibited virus-dependent activation of Rho A and Rac1 GTPases, as well as phosphorylation of caveolin-1. In addition, JEV entry induced the clustering of lipid rafts. MβCD or filipin III treatment resulted in a marked inhibition of viral internalization, and JEV-induced phosphorylation of EGFR and the activation of downstream PI3 K signaling were almost completely inhibited.Conclusion: EGFR-PI3 K signaling pathway initiated by virus attachment induced Rho GTPase activation and led to subsequent events that promoted caveolin-1-mediated virus endocytosis. lipid rafts function as platforms for JEV-induced signal transduction.SummaryViruses always utilize the cellular mechanism of endocytosis to establish their infectious entry into permissive cells; endocytosis allows viruses gain access to viral replication sites by overcoming the obstacles, such as the plasma membrane, crowded cytoplasm, and helps viruses to overcome host immune surveillance by leaving minimal evidence on the cell surface. In this study, a targeted si RNA screening platform assay was established to identify and analyze key cellular genes and pathways utilized for JEV entry process. Our findings show that:(1) JEV deploys a caveolin-1-mediated and dynamin-dependent endocytic mechanism to entry into human neuronal cells.(2) The endocytosis process required a two-step regulation of the actin cytoskeleton via activation of Rho A and Rac1. Virus-induced Rho A activation promoted the phosphorylation and aggregation of caveolin-1 on cell membranes, and then phosphorylated caveolin-1 activated Rac1, leading to endocytosis of virus within caveolin-1-associated vesicles.(3) EGFR-PI3 K signaling is an active participant in mediating Rho GTPase activation and subsequent signaling events that are required for caveolin-1-mediated virus endocytosis.(4) After internalization, JEV required microtubule-dependent trafficking and transport to Rab5-positive endosomes before the release of viral genome into the cytoplasm, resulting in productive infection. These findings allow us to propose a schematic diagram depicting the signals and endocytic pathways required for JEV entry into human neuronal cells...