Japanese encephalitis virus manipulates lysosomes membrane for RNA replication and utilizes autophagy components for intracellular growth

https://doi.org/10.1016/j.vetmic.2021.109025Get rights and content

Highlights

  • Viral genome replication took place in lysosomes, not in autophagosomes.

  • Viral RNAs strongly colocalized with LC3 and LAMP1.

  • Autophagy pathway was activated and maintained an intact fusion process.

  • Deletion of autophagy impeded the early replicative events.

  • Fusion of autophagosome-lysosome affected post-RNA replication processes.

Abstract

Japanese encephalitis virus is absolutely dependent on their host cells and has evolved various strategies to manipulate the cellular secretory pathways for viral replication. However, how cellular secretory pathways are hijacked, and the origin of the viral vesicles remains elusive during JEV replication. Here we show how JEV manipulates multiple components of the cellular secretory pathway, including autophagic machinery, to generate a superior environment for genome replication. We utilized double-strand RNA antibodies to label JEV RNA complex seeking the viral replication compartments and found that JEV genome replication takes place in lysosomes (LAMP1), not in autophagosomes (LC3). Subsequently, in situ hybridization results showed that viral RNAs (vRNAs) of JEV strongly colocalized with LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that autophagy plays an active role in JEV replication. Interestingly, we found that JEV utilized autophagic components for intracellular growth in an autophagy-dependent manner and the fusion of autophagosome-lysosome plays a positive role in JEV post-RNA replication processes. Collectively, our findings demonstrate that JEV can manipulate cellular secretory pathway to form genome replication organelles and exploit autophagy components for intracellular growth, providing new insights into the life cycle of JEV and uncovering an attractive target for antiviral drugs.

Introduction

Japanese encephalitis virus (JEV), a member of the genus Flavivirus (Fernandez-Garcia et al., 2009), is an important human and animal pathogen that causes a wide spectrum of clinical manifestations, including mild febrile illness and irreversible neurological damage (Basumatary et al., 2013). JEV is a positive-sense RNA enveloped virus that contains a ∼11 kb positive-sense RNA genome encoding three structural proteins and seven nonstructural proteins (Yun and Lee, 2018). JE is most prevalent in eastern and southern Asia causing a fatality rate of 25 % in humans (Wang and Liang, 2015).

Being obligate pathogens, JEV and other RNA viruses exploit and hijack host cell machinery and functions to facilitate viral infection and produce progeny viruses (Miller and Krijnse-Locker, 2008). The secretory pathway exists in all eukaryotic cells and is a fundamental process facilitated by diverse highly conserved molecules and mechanism mediating movement of proteins between secretory organelles and membrane-bound transport intermediates (Sager et al., 2018). Previous study demonstrated that the compartments of the secretory pathway contain endoplasmic reticulum (ER), ER exit sites (ERES), the ER-to-Golgi intermediate compartment (ERGIC), the Golgi complex, the cis-Golgi network (CGN) and the trans-Golgi network (TGN) (Behnia and Munro, 2005). Distinct steps of the virus life cycle occur in association with the secretory pathway and intracellular membranes, which is often modified to complete their infection, replication, assembly and release (Robinson et al., 2018). JEV enters host cells through clathrin-dependent endocytosis and is transported from early to recycling endosomes before the release of nucleocapsid into the cytoplasm (Liu et al., 2017). Replication membranes appear to originate from the ER during the genome replication of picornaviruses, flaviviruses and coronaviruses (Schlegel et al., 1996), while the origin of replication membranes for togaviruses may come from the endosomes/lysosomes and mitochondria (Magliano et al., 1998). However, studies on how JEV manipulates the cellular secretory pathways for viral replication are rare.

As a crucial component of the secretory pathway, autophagy plays an important role in maintaining homeostasis by degrading misfolded protein and damaged organelles (Levine and Kroemer, 2019). The autophagy pathway is a highly conserved cellular feedback that can be induced by diverse conditions, such as nutrient deprivation, pathogen invasion and other stresses (Kroemer et al., 2010). Canonical autophagy undergoes initiation and nucleation of phagophore, phagophore elongation and closure, and autophagosome-lysosome fusion and cargo degradation, three basic processes performed by a series of autophagy gene products (Galluzzi et al., 2017). The main lipid sources of autophagosomes are thought to generated from multiple cellular structures, such as the ER (Hayashi-Nishino et al., 2009) and the mitochondrial membrane (Hailey et al., 2010). Not surprisingly, multiple components of the molecular machinery for autophagy may mediate cellular membrane rearrangement and vesicle trafficking during the formation of autophagosomes. Increasing evidence suggests that various viruses can interact with the autophagy pathway to bring antiviral or proviral functions, even during the same viral infection (Abernathy et al., 2019). It has been reported that JEV replication can be negatively regulated by autophagy and its replication machinery is localized in LC3-I and EDEMI-containing membranes (Sharma et al., 2014), while other contrasting study shows that JEV manipulates autophagy to evade the immune system by suppressing type I IFN production (Jin et al., 2013). Therefore, key concerns are to learn which secretory organelle JEV reorganizes as an RNA replication platform and how autophagy is involved in JEV’s life cycle.

Currently, the study of flavivirus-host cell interactions has advanced greatly, but it is still unclear how JEV hijacks the cellular secretory pathways, including autophagic machinery, to gear up for replication. To determine which component of the cellular secretory pathway is exploited by JEV, immunofluorescence assay was performed to explore the colocalization of JEV dsRNA (an obligatory intermediate of positive-strand RNA virus replication) with secretory organelles. We found that JEV’s replication compartments originate from ER, ERGIC and lysosomes, not from autophagosomes. In situ hybridization results also discovered that JEV vRNAs markedly colocalized with calnexin, ERGIC53 and LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that although the autophagosome does not provide the replication structure, autophagy plays an active role in JEV replication. Then, we found that the depletion of ATG5, ATG16L1, ATG12, and LC3B significantly inhibited RNA replication, NS1expression and mature virion production during its early replicative step. JEV replication also be significantly inhibited by chloroquine treatment, but its effects occur in JEV post-RNA replication processes. This study highlights novel insight into the host secretory pathway, including the autophagy pathway, when seeking to understand how JEV hijacks and manipulates this pathway for purposes of viral replication and potential drug targets.

Section snippets

Cells and virus

HeLa cells, HEK293 T cells and BHK21 cells were cultured in DMEM (Gibco) with 10 % fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. Japanese encephalitis virus (SA14 strain) was reserved in the College of Veterinary Medicine, South China Agricultural University.

Confocal microscopy

HeLa cells were seeded on coverslips in 24-well plates and infected with JEV at an MOI (multiple of infection) of 20 for 12 h. The cells were fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 15 min

JEV RNA machinery is localized in ER, ERGIC and lysosomes

JEV is an enveloped virus that matures intracellularly and double-stranded RNA as an intermediate product of RNA genome replication is synthesized. To provide a characterization of the viral RNA replication membranes in cells infected with JEV, we targeted dsRNA by using J2 antibody as a marker. In the early stages of JEV infection, we performed an immunofluorescence assay to explore the colocalization between dsRNA with secretory organelles markers, including early endosome (EEA1), late

Discussion

JEV and other flaviviruses are enveloped viruses that share similar morphological and functional properties with transport vesicles of the host cell, which provides a convenient condition for the manipulation of intracellular secretory pathway (Thaa et al., 2010). Here, we focus on the point that JEV exploits host secretory pathway, including autophagic machinery, to facilitate its viral replication. In our study, we found that LAMP1 (lysosomes), EGFP-ERGIC53 (ERGIC) and calnexin (ER) these

Declaration of Competing Interest

The author(s) declare that they have no conflict of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31830097, 31672586), the Key Research and Development Program of Guangdong Province (2019B020218004), Earmarked Found for China Agriculture Research System (CARS-41-G16), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2018, Wenbao Qi), and Young Scholars of Yangtze River Scholar Professor Program (2019, Wenbao Qi).

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