GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

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Faculty Opinions recommendation of GRP78 is an important host factor for Japanese encephalitis virus entry and replication in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727169751.793567004 ◽

2019 ◽

Author(s):

Arup Banerjee

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Mammalian Cells ◽

Virus Entry ◽

Host Factor ◽

Encephalitis Virus ◽

Important Host

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RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication

Journal of Virology ◽

10.1128/jvi.01306-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 2

Author(s):

Yi Wang ◽

Xiaoyan Chen ◽

Jiong Xie ◽

Shili Zhou ◽

Yanxia Huang ◽

...

Keyword(s):

Life Cycle ◽

Dengue Virus ◽

Zika Virus ◽

Rna Helicase ◽

Host Factor ◽

Host Factors ◽

Helicase Activity ◽

Nonstructural Proteins ◽

Rna Helicase A ◽

Important Host

ABSTRACT Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents. IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

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Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

Journal of Virology ◽

10.1128/jvi.00197-18 ◽

2018 ◽

Vol 92 (12) ◽

Cited By ~ 10

Author(s):

Le Ma ◽

Fang Li ◽

Jing-Wei Zhang ◽

Wei Li ◽

Dong-Ming Zhao ◽

...

Keyword(s):

Life Cycle ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Virus Assembly ◽

Rna Replication ◽

Protein Processing ◽

Host Factor ◽

Transmembrane Domains ◽

Cell Entry ◽

Antiviral Targets

ABSTRACTSignal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1–49) and NS2B(84–131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1–49) and P112A in NS2B(84–131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91–169), which contains two transmembrane domains, was involved in interactions with both NS2B(1–49) and NS2B(84–131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions.IMPORTANCEUnderstanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

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Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle

Journal of Virology ◽

10.1128/jvi.02336-20 ◽

2021 ◽

Author(s):

Sapna Sehrawat ◽

Renu Khasa ◽

Arundhati Deb ◽

Surendra Kumar Prajapat ◽

Suvadip Mallick ◽

...

Keyword(s):

Life Cycle ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Host Factor ◽

Encephalitis Virus ◽

Host Factors ◽

Viral Rna ◽

High Morbidity ◽

Replication Complex ◽

Virus Life Cycle

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle – nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses. Importance JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

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mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes

Journal of Virology ◽

10.1128/jvi.01348-16 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 13

Author(s):

Ke Liu ◽

Yingjuan Qian ◽

Yong-Sam Jung ◽

Bin Zhou ◽

Ruibing Cao ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Envelope Protein ◽

Viral Encephalitis ◽

Encephalitis Virus ◽

Content Type ◽

Flavivirus Infection ◽

New Strategies

ABSTRACT Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti. The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro. mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature. IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7–JEV complex to facilitate virus infection in vivo and in vitro. Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

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USP38 Inhibits Zika Virus Infection by Removing Envelope Protein Ubiquitination

10.20944/preprints202107.0634.v1 ◽

2021 ◽

Author(s):

Yingchong Wang ◽

Qi Li ◽

Dingwen Hu ◽

Daolong Gao ◽

Wenbiao Wang ◽

...

Keyword(s):

Zika Virus ◽

Envelope Protein ◽

Host Protein ◽

South American ◽

Zikv Infection ◽

Potential Therapeutic Target ◽

Treatment And Prevention ◽

Protein Ubiquitination ◽

Neurologic Disorders ◽

Novel Host

Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South American has caused severe human congenital and neurologic disorders. Thus, it is vitally important to figure out inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost ability to inhibit the infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection.

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USP38 Inhibits Zika Virus Infection by Removing Envelope Protein Ubiquitination

Viruses ◽

10.3390/v13102029 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2029

Author(s):

Yingchong Wang ◽

Qin Li ◽

Dingwen Hu ◽

Daolong Gao ◽

Wenbiao Wang ◽

...

Keyword(s):

Host Resistance ◽

Zika Virus ◽

Envelope Protein ◽

Host Protein ◽

Zikv Infection ◽

Potential Therapeutic Target ◽

Treatment And Prevention ◽

Protein Ubiquitination ◽

Neurologic Disorders ◽

Novel Host

Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South America has caused severe human congenital and neurologic disorders. Thus, it is vitally important to determine the inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to the ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost the ability to inhibit infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection.

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TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection

Viruses ◽

10.3390/v10110630 ◽

2018 ◽

Vol 10 (11) ◽

pp. 630 ◽

Cited By ~ 9

Author(s):

Jichen Niu ◽

Ya Jiang ◽

Hao Xu ◽

Changjing Zhao ◽

Guodong Zhou ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Point Mutation ◽

Japanese Encephalitis ◽

Virus Entry ◽

A549 Cells ◽

Binding Pocket ◽

Cytoplasmic Domain ◽

Encephalitis Virus ◽

Host Cells ◽

Attachment Factor

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.

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An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles

Viruses ◽

10.3390/v13091727 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1727

Author(s):

Iris Medits ◽

Franz X. Heinz ◽

Karin Stiasny

Keyword(s):

Life Cycle ◽

Envelope Protein ◽

Transmembrane Domain ◽

Viral Life Cycle ◽

Compensatory Mutation ◽

Sequence Element ◽

Conserved Sequence ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Stable Particles

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called “stem” region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

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