Japanese encephalitis virus restricts HMGB1 expression to maintain MAPK pathway activation for viral replication

Highlights

• JEV infection down-regulates HMGB1 expression.

• HMGB1 modulates JEV replication.

• HMGB1 suppressed MAPK pathway.

• The weakening of MAPK pathway negatively regulates JEV infection.

• HMGB1 exerts negative modulation of JEV replication through the MAPK pathway.

Abstract

Japanese encephalitis virus (JEV) is a typical insect-borne flavivirus and an important zoonotic pathogen that causes human viral encephalitis and reproductive failure in pigs. Various strategies were utilized by JEV to facilitate its replication. It is important to identify key molecules that mediate JEV infection, as well as to investigate their underlying mechanism. In this study, the critical role of high-mobility group box 1 (HMGB1), a non-histone, DNA-binding protein, was assessed in JEV propagation. Upon JEV infection, the HMGB1 mRNA and protein levels were down-regulated at late infection in Huh7 cells. JEV replication was significantly enhanced with HMGB1 knock-down by siRNA and knock-out by the CRISPR/Cas9 system, whereas JEV growth was restricted in HMGB1-over-expressed Huh7 cells. Further investigation showed that HMGB1 suppressed MAPK pathway, and demonstrated that the weakening of MAPK pathway negatively regulated JEV infection. Together, these results suggested that JEV restricted HMGB1 expression to maintain MAPK pathway activation for viral replication. Our data showed that HMGB1 played a key role in JEV infection, providing the potential for the development of a novel drug to combat JEV infection.

Introduction

Japanese encephalitis, an infectious central nervous system disease, is caused by Japanese encephalitis virus (JEV). JEV belongs to the genus Flavivirus in the family Flaviviridae, and it causes human viral encephalitis and reproductive failure in pigs. JEV transmission occurs from Southeast Asia to the western Pacific islands (Solomon, 2000). Recently, mounting evidence indicates that JEV has been detected in non-endemic areas, such as Italy and the Tibet Autonomous Region (a high-altitude area) in China, increasing the risk of public exposure (Li et al., 2011; Ravanini et al., 2012; Zhang et al., 2017). In Asia, the genotype III (GIII) virus was gradually replaced by the genotype I (GI) virus, thus becoming a mainstream genotype (Gao et al., 2013), but current vaccines using GIII virus strains do not provide sufficient protection against the GI virus (Fan et al., 2012, 2013). Additionally, specific drug treatments to inhibit JEV are not available. Therefore, the discovery of cellular molecules that mediate JEV infection is urgent.

JEV is a single-stranded positive-sense RNA virus consisting of three structure proteins (capsid, envelope, and pre-membrane) and seven non-structure proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Sumiyoshi et al., 1987). The viral life cycle cannot be completed without the involvement of host proteins (Mahon et al., 2014). Viruses utilize the functions of cellular factors to establish effective infection (Zakaria et al., 2018). High mobility group box 1 (HMGB1), a non-histone, DNA-binding protein, is a highly conserved protein belonging to the high-mobility group box (HMGB) protein family (Lotze and Tracey, 2005). HMGB1 in the nucleus is responsible for many DNA activity-associated functions (e.g., DNA replication, transcription, and repair). In addition to functions in the nucleus, HMGB1 plays a significant role in inflammation, immunity, as well as cell growth and death (Kang et al., 2014).

There is accumulating evidence that HMGB1 modulates viral infections. Nuclear HMGB1 binds to the promoter region of the ISG to enhance its transcription and exert an anti-DENV effect (Zainal et al., 2017). Porcine circovirus type 2 (PCV2) can trigger HMGB1 translocation from nucleus to cytoplasm, which promotes viral replication (Sun et al., 2020). Influenza virus replication and the activity of the viral polymerase are positively regulated by the interaction between nuclear HMGB1 and nucleoprotein (Moisy et al., 2012). The release of extracellular HMGB1 from hepatitis virus C (HCV) infection inhibits secondary infection by the virus (Jung et al., 2011). HMGB1 can interact with the HCV 5′untranslated region to enhance viral replication (Yu et al., 2016). In addition, HMGB1 is involved in the mitogen-activated protein kinases (MAPK) signaling pathway in porcine epidemic diarrhea virus (PEDV) and Newcastle disease virus (NDV) infections (Qu et al., 2018; Gao et al., 2020).

The MAPK pathways are important signal transducing enzymes in eukaryotes that regulate cell proliferation and death (Chang and Karin, 2001). Extracellular signal-regulated kinases (ERK), MAPK p38, and c-Jun N-terminal kinases (JNK) are the three most frequently studied kinases. The MAPK cascade pathways play important roles in viral infection. Foot-and-mouth disease virus (FMDV) and influenza virus infections activate MAPK pathway to enhance viral replication (Yu et al., 2019; Zhu et al., 2020).

In this study, we mainly investigated the effect of HMGB1 on JEV replication. Intriguingly, we found that JEV modulated HMGB1 to activate MAPK pathway, which facilitated viral replication. These findings indicated that HMGB1 played a crucial role in JEV infection, providing the potential for the development of a novel drug to combat JEV infection.