Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells

Highlights

• JEV can infect neuronal cells through clathrin-independent endocytosis.

• Actin filament organization proteins are crucial for JEV infection in neuronal cells.

• Small molecule inhibitors CK-548 and Wiskostatin inhibit JEV infection.

• Proteins ATG12, BECN1, VAPA, VAPB and VCP are host-factors for JEV replication.

Abstract

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

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.