Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells
Introduction
Virus-host interaction involves a series of complex molecular and signaling events that are largely determined by the host cell lineage [[1], [2], [3], [4], [5]]. For several enveloped viruses, infection initiates with binding mediated by attachment factors and specific receptors, followed by internalization into one of the several endocytic portals operating at the plasma membrane [[6], [7], [8], [9], [10]]. Understanding the membrane trafficking requirements for pathogenic viruses is crucial for rational design of anti-virals [[11], [12], [13]].
Japanese encephalitis virus (JEV), a neurotropic flavivirus is a leading global cause of viral encephalitis with high morbidity and mortality [[14], [15], [16]]. Most flaviviruses adopt clathrin mediated endocytosis (CME) for establishing infection [8,[17], [18], [19]], however independent studies have now conclusively established that while JEV uses CME to enter epithelial cells and fibroblasts [[20], [21], [22], [23]], it infects neuronal cells through clathrin-independent endocytosis (CIE) [21,24,25]. The CIE of JEV in mouse neuronal cells was found to require dynamin, cholestrol, Rab5 and a dynamic actin network [21], while CIE in rat neuronal cells required dynamin and caveolin-1 [24]. RNA interference based studies have demonstrated that JEV replication in the human neuronal cell line SK-N-SH required caveolin-1, along with RHOA and RAC1 mediated actin cytoskeleton rearrangements [25]. Another recent study in human brain microvascular endothelial cells (HBMEC) has reconfirmed a role for CIE, with an essential role for Src and ezrin-mediated actin cytoskeleton polymerization for JEV entry [26].
Studies from our lab based on RNA interference screening of membrane trafficking genes in the epithelial cell line HeLa showed that JEV depends extensively on components of CME pathway for establishing infection, along with the members of the ARP2/3 complex [23]. Here we have utilized the same library to identify membrane trafficking host-factors essential for JEV replication in the human neuronal cell line IMR-32. The Rho family GTPAse RAC1 formed a central node with proteins of the two actin based processes playing a crucial role-the ARP2/3 complex and N-WASP family; and the PAK1-ROCK-2-LIMK1 signaling complex. We also identify other common host factors: ATG12, BECN1, VAPA, VAPB & VCP for JEV infection across epithelial and neuronal cell lineages.
Section snippets
Cells and virus
Human neuronal cell line IMR-32, and porcine stable kidney cell line- PS were obtained from National Centre for Cell Science (NCCS), Pune, India. SH-SY5Y and HeLa (CCL-2) cell lines were obtained from ATCC. IMR-32, SH-SY5Y and HeLa cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM), and PS cells in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 IU/ml Penicillin, 100 μg/ml Streptomycin and 2 mM l-Glutamine (PSG) solution. JEV strain
Establishment of siRNA screen in human neuronal IMR-32 cells
As a first step, we optimised the siRNA transfection conditions in IMR-32 cells to achieve maximum transfection efficiency. This was established by co-transfection of the non-targeting (NT) siRNA, or clathrin light chain (CLTB) siRNA with siGLO Green transfection indicator, which is a fluorescent oligonucleotide duplex that localizes to the nucleus (Fig. 1a). Fluorescence quantification of siGLO indicated that a transfection efficiency of 80–90% was achieved in IMR-32 cells (Fig. 1a).
We also
Discussion
Membrane trafficking networks are involved at multiple steps of the virus life-cycle ranging from entry to egress [6,9,54]. Most flaviviruses including JEV, have been shown to exploit a CME pathway to establish infection [8,[17], [18], [19]]. However, studies in the past ten years have shown that several viruses can opt for more than one route for endocytosis, depending on the cell type, which could be potentially caused by different receptor usage and/or activation of specific signalling
Funding information
This work was supported by DBT grant BT/MB/01/VIDRC/08 and by DBT intra-mural research funds to THSTI & RCB. RK was supported by ICMR-SRF fellowship, and was a PhD scholar with Symbiosis International (Deemed University), Symbiosis Knowledge Village, Gram Lavale, Mulshi Taluka, Pune, Maharashtra 412115, India. PS is supported by UGC-JRF fellowship.
Author statement
The authors declare that they have no known competing financial interest or personal relationship that influenced this study.
Declaration of competing interest
None.
Acknowledgements
We all thank all members of the Virology labs in THSTI & RCB.
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Virus-host Interactions in Early Japanese Encephalitis Virus Infection
2023, Virus ResearchPathobiology of Japanese encephalitis virus infection
2021, Molecular Aspects of MedicineCitation Excerpt :The deployment of a differential endocytic pathway in neuronal cells is likely to be governed by receptor usage. RNA interference-based screens have also identified several proteins involved in actin dynamics and other key host factors such as ezrin, platelet-derived growth factor receptor beta, and valosin-containing protein that are essential for virus replication (Khasa et al., 2019, 2020; Xu et al., 2016; Liu et al., 2020a; Sehrawat, 2021; Zhou et al., 2021). Similarly, CRISPR screening using a porcine sgRNA library also identified several critical host factors associated with JEV replication in porcine cells (Zhao et al., 2020).
Japanese Encephalitis Virus-Infected Cells
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2021, Journal of Virology
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Present address: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.