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A Metabolomics Approach to Unravel Cricket Paralysis Virus Infection in Silkworm Bm5 Cells.

ABSTRACT: How a host metabolism responds to infection with insect viruses and how it relates to pathogenesis, is little investigated. Our previous study observed that Cricket paralysis virus (CrPV, Dicistroviridae) causes short term persistence in silkworm Bm5 cells before proceeding to acute infection. In this study, a metabolomics approach based on high resolution mass spectrometry was applied to investigate how a host metabolism is altered during the course of CrPV infection in Bm5 cells and which changes are characteristic for the transition from persistence to pathogenicity. We observed that CrPV infection led to significant and stage-specific metabolic changes in Bm5 cells. Differential metabolites abundance and pathway analysis further identified specific metabolic features at different stages in the viral life cycle. Notably, both glucose and glutamine levels significantly increased during CrPV persistent infection followed by a steep decrease during the pathogenic stages, suggesting that the central carbon metabolism was significantly modified during CrPV infection in Bm5 cells. In addition, dynamic changes in levels of polyamines were detected. Taken together, this study characterized for the first time the metabolic dynamics of CrPV infection in insect cells, proposing a central role for the regulation of both amino acid and carbohydrate metabolism during the period of persistent infection of CrPV in Bm5 cells.

SUBMITTER: Wang LL

PROVIDER: S-EPMC6784103 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A Metabolomics Approach to Unravel Cricket Paralysis Virus Infection in Silkworm Bm5 Cells.

Wang Luo-Luo LL Swevers Luc L Rombouts Caroline C Meeus Ivan I Van Meulebroek Lieven L Vanhaecke Lynn L Smagghe Guy G

Viruses 20190916 9

How a host metabolism responds to infection with insect viruses and how it relates to pathogenesis, is little investigated. Our previous study observed that Cricket paralysis virus (CrPV, Dicistroviridae) causes short term persistence in silkworm Bm5 cells before proceeding to acute infection. In this study, a metabolomics approach based on high resolution mass spectrometry was applied to investigate how a host metabolism is altered during the course of CrPV infection in Bm5 cells ...[more]

PMID: 31527462

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Project description:UnlabelledDicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells.ImportanceDicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA into Drosophila cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5' untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.

Project description:Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

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A Metabolomics Approach to Unravel Cricket Paralysis Virus Infection in Silkworm Bm5 Cells.

Publications

A Metabolomics Approach to Unravel <i>Cricket Paralysis Virus</i> Infection in Silkworm Bm5 Cells.

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