Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Sharma, Manish; Bhattacharyya, Sankar; Nain, Minu; Kaur, Manpreet; Sood, Vikas; Gupta, Vishal; Khasa, Renu; Abdin, M. Z.; Vrati, Sudhanshu; Kalia, Manjula

doi:10.4161/auto.29455

Cited by 94 publications

(106 citation statements)

References 74 publications

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“…Previous studies in our laboratory have shown that virus replication complexes are established in the infected cell by 6 to 8 h p.i. (41,42). They are detectable as discrete foci of dsRNA and viral nonstructural proteins, like NS1, NS3, and NS5, by immunofluorescence (42).…”

Section: Discussionmentioning

confidence: 99%

“…Subtilase cytotoxin cleavage of GRP78 also activates the ER stress pathways (68,69) and facilitates ER-associated degradation (ERAD) (70). Since ER stress pathways and ERAD are closely associated with JEV replication (42,67,71), it is also possible that the effect of GRP78 cleavage on JEV replication is manifested by dysregulation of these cellular pathways. The multiple critical roles of GRP78 in the infectious life cycle of JEV highlight the potential of this host protein as a novel target for antiviral development against JEV.…”

Section: Discussionmentioning

confidence: 99%

“…(41,42). They are detectable as discrete foci of dsRNA and viral nonstructural proteins, like NS1, NS3, and NS5, by immunofluorescence (42). Inhibition of virus replication by the addition of SubAB toxin at 6 and 12 h p.i.…”

Section: Discussionmentioning

confidence: 99%

“…Horseradish peroxidase (HRP)-coupled secondary antibodies were obtained from Jackson Immunochemicals. Rabbit polyclonal antibodies against JEV NS1 and NS3 were produced in the laboratory and have been described previously (42). All the JEV antibodies used in the study reacted with proteins of the expected sizes on Western blotting in JEV-infected cells, but not in mock-infected cells.…”

Section: Methodsmentioning

confidence: 99%

“…Virus replication is detectable in cells by 6 to 8 h p.i., as seen from double-stranded RNA (dsRNA) foci (41). These dsRNA puncta show extensive colocalization with JEV NS1, NS3, and NS5 nonstructural proteins (41,42). To test if GRP78 had any role in the formation of the replication complex, we performed immunofluorescence staining of dsRNA and NS3 in JEV-infected Neuro2a cells treated with wild-type or mutant toxin at 6 h p.i.…”

Section: Figmentioning

confidence: 99%

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

Nain

Mukherjee

Karmakar

et al. 2017

J Virol

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 3 more Smart Citations

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

Nain

Mukherjee

Karmakar

et al. 2017

J Virol

Self Cite

116

113

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The interplay between pathogens and Atg8 family proteins: thousand‐faced interactions

2021

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Autophagy is an intracellular degradation and recycling process that can also remove pathogenic intracellular bacteria and viruses from within cells (referred to as xenophagy) and activate the adaptive immune responses. But autophagy—especially Atg proteins including Atg8 family members—can also have proviral and probacterial effects. In this review, we summarize known interactions of bacterial, parasitic, and viral proteins with Atg8 family proteins and the outcome of these interactions on pathogen replication, autophagy, or mitophagy. We discuss the value of prediction software and the research methodology in the study of pathogen protein‐Atg8 family protein interactions, with selected examples of potential LC3‐interacting region motif‐containing SARS‐CoV‐2 proteins.

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Demarcation of Viral Shelters Results in Destruction by Membranolytic GTPases: Antiviral Function of Autophagy Proteins and Interferon‐Inducible GTPases

et al. 2018

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A hallmark of positive-sense RNA viruses is the formation of membranous shelters for safe replication in the cytoplasm. Once considered invisible to the immune system, these viral shelters are now found to be antagonized through the cooperation of autophagy proteins and anti-microbial GTPases. This coordinated effort of autophagy proteins guiding GTPases functions against not only the shelters of viruses but also cytoplasmic vacuoles containing bacteria or protozoa, suggesting a broad immune-defense mechanism against disparate vacuolar pathogens. Fundamental questions regarding this process remain: how the host recognizes these membranous structures as a target, how the autophagy proteins bring the GTPases to the shelters, and how the recruited GTPases disrupt these shelters. In this review we discuss these questions, the answers to which will significantly advance our understanding of the response to vacuole-like structures of pathogens, thereby paving the way for the development of broadly effective anti-microbial strategies for public health.

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Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Cited by 94 publications

References 74 publications

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

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

The interplay between pathogens and Atg8 family proteins: thousand‐faced interactions

Demarcation of Viral Shelters Results in Destruction by Membranolytic GTPases: Antiviral Function of Autophagy Proteins and Interferon‐Inducible GTPases

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