Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation

ABSTRACTViruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV’s preference of glutamine to glucose rather than glutamine’s superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCEViruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

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Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway

Viruses ◽

10.3390/v12050499 ◽

2020 ◽

Vol 12 (5) ◽

pp. 499 ◽

Cited By ~ 2

Author(s):

Shelby Powell Kesterson ◽

Jeffery Ringiesn ◽

Vikram N. Vakharia ◽

Brian S. Shepherd ◽

Douglas W. Leaman ◽

...

Keyword(s):

Protein Synthesis ◽

Viral Protein ◽

Molecular Mechanisms ◽

Freshwater Ecosystems ◽

Innate Immune ◽

Host Cells ◽

Initiation Factor ◽

Viral Hemorrhagic Septicemia Virus ◽

Viral Protein Synthesis ◽

Viral Hemorrhagic Septicemia

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

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Bortezomib inhibits chikungunya virus replication by interfering with viral protein synthesis

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008336 ◽

2020 ◽

Vol 14 (5) ◽

pp. e0008336

Author(s):

Parveen Kaur ◽

Laura Sandra Lello ◽

Age Utt ◽

Sujit Krishna Dutta ◽

Andres Merits ◽

...

Keyword(s):

Protein Synthesis ◽

Virus Replication ◽

Viral Protein ◽

Chikungunya Virus ◽

Viral Protein Synthesis

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Effects of Traditional Kampo Drugs and Their Constituent Crude Drugs on Influenza Virus Replication In Vitro: Suppression of Viral Protein Synthesis by Glycyrrhizae Radix

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/3230906 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Toshihito Nomura ◽

Masaya Fukushi ◽

Kosuke Oda ◽

Akifumi Higashiura ◽

Takashi Irie ◽

...

Keyword(s):

Protein Synthesis ◽

Influenza Virus ◽

Virus Replication ◽

Viral Protein ◽

Culture Media ◽

Virus Infectivity ◽

Viral Protein Synthesis ◽

Glycyrrhizae Radix ◽

Influenza Virus Replication ◽

Crude Drugs

An influenza virus epidemic is an important issue in public hygiene, and continuous development on an effective drug is required. Kampo medicine is a traditional medicine that is used clinically for treatment of various diseases in Japan and other East Asian countries. We evaluated the effects of the Kampo drugs maoto, kakkonto, senkyuchachosan, jinkokato, and bakumondoto, which are prescribed for treatment of respiratory symptoms including symptoms caused by influenza, on influenza virus replication in cultured cells. Culture media of influenza virus-infected MDCK(+) cells were tested for hemagglutination and infectivity at 24 h after the addition of Kampo drugs at various concentrations, and four of the five Kampo drugs were found to inhibit virus release to the culture media. These drugs inactivated virus infectivity not by acting on virus particles but by acting on virus-infected cells. In addition, when six crude drugs (Atractylodis lanceae rhizome, Citri unshiu pericarpium, Cnidii rhizome, Glycyrrhizae radix, Rehmanniae radix, and Saposhnikoviae radix) that constitute the effective Kampo drugs were examined, the strongest activity was found for Glycyrrhizae radix (IC50 = 0.27 mg/ml), which selectively suppressed viral protein synthesis. Since Glycyrrhizae radix is contained in many Kampo drugs, it may give anti-influenza virus activity to a broad range of Kampo drugs.

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Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis

Journal of Virology ◽

10.1128/jvi.01891-07 ◽

2007 ◽

Vol 82 (2) ◽

pp. 840-848 ◽

Cited By ~ 63

Author(s):

Ping Zhang ◽

Bertram L. Jacobs ◽

Charles E. Samuel

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Vaccinia Virus ◽

Hela Cells ◽

Viral Protein ◽

Deletion Mutant ◽

Mutant Phenotype ◽

Viral Protein Synthesis ◽

Virus Growth ◽

Α Subunit

ABSTRACT The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log10 in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

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The Ubiquitin-Proteasome System Is Necessary for Efficient Replication of Human Astrovirus

Journal of Virology ◽

10.1128/jvi.01809-17 ◽

2017 ◽

Vol 92 (2) ◽

Cited By ~ 2

Author(s):

Luis A. Casorla-Pérez ◽

Tomás López ◽

Susana López ◽

Carlos F. Arias

Keyword(s):

Protein Synthesis ◽

Viral Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Progeny Production ◽

Viral Protein Synthesis ◽

Viral Progeny ◽

Efficient Replication ◽

Ubiquitin Proteasome ◽

Human Astrovirus

ABSTRACT Astroviruses, members of the family Astroviridae, represent an important cause of human gastroenteritis in the world. The cellular factors required for astrovirus replication have been poorly studied. In this work, we evaluated the relevance of the ubiquitin-proteasome system (UPS) in the replication of Yuc8, a human astrovirus serotype 8 strain. We found that proteasome inhibitors decrease the production of infectious viral progeny at a step in the replication cycle subsequent to virus entry. The inhibition of proteasome activity decreases viral RNA levels and viral protein synthesis; similarly, the inhibition of ubiquitination by chemical inhibitors or RNA interference (RNAi) reduces the production of viral progeny as well as viral protein synthesis. The effect on viral progeny production induced by proteasome inhibitors is not explained by a reduction in the pool of monoubiquitin or the induction of early apoptosis or autophagy. Our observations are consistent with the need of the proteolytic activity of the UPS for the efficient replication of the virus and suggest that UPS is necessary for the production of genomic and subgenomic RNA but not for antigenomic RNA. IMPORTANCE Astroviruses are a major cause of gastroenteritis in young humans and animals, and recently, it was associated with fatal encephalitis in humans. The role of the ubiquitin-proteasome system in the replication of these viruses has not been studied previously. In this work, we present evidence that supports that the proteolytic activity of the proteasome is necessary for efficient viral progeny production and that this proteolytic system is required for the accumulation of both genomic and subgenomic viral RNAs.

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