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A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junin Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

ABSTRACT: Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2?. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2?, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2?, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2? phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

SUBMITTER: King BR

PROVIDER: S-EPMC5512243 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

King Benjamin R BR Hershkowitz Dylan D Eisenhauer Philip L PL Weir Marion E ME Ziegler Christopher M CM Russo Joanne J Bruce Emily A EA Ballif Bryan A BA Botten Jason J

Journal of virology 20170712 15

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) ...[more]

PMID: 28539447

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Project description:UnlabelledThe Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. This work provides immunofluorescence evidence that, unlike those of New World arenaviruses, the replication and transcription complexes (RTC) of lymphocytic choriomeningitis virus (LCMV) colocalize with eukaryotic initiation factor 4E (eIF4E) and that eIF4E may participate in the translation of LCMV mRNA. Additionally, we identify two residues in the LCMV nucleoprotein (NP) that are conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, Y125, was confirmed to be phosphorylated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). NP Y125 is located in the N-terminal region of NP that is disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence analysis demonstrated that NP T206 is required for the formation of the punctate RTC that are typically observed during LCMV infection. A minigenome reporter assay using NP mutants, as well as Northern blot analysis, demonstrated that although NP T206A does not form punctate RTC, it can transcribe and replicate a minigenome. However, in the presence of matrix protein (Z) and glycoprotein (GP), translation of the minigenome message with NP T206A was inhibited, suggesting that punctate RTC formation is required to regulate viral replication. Together, these results highlight a significant difference between New and Old World arenaviruses and demonstrate the importance of RTC formation and translation priming in RTC for Old World arenaviruses.ImportanceSeveral members of the Arenaviridae cause hemorrhagic fevers and are classified as category A pathogens. Arenavirus replication-transcription complexes (RTC) are nucleated by the viral nucleoprotein. This study demonstrates that the formation of these complexes is required for virus viability and suggests that RTC nucleation is regulated by the phosphorylation of a single nucleoprotein residue. This work adds to the body of knowledge about how these key viral structures are formed and participate in virus replication. Additionally, the fact that Old World arenavirus complexes colocalize with the eukaryotic initiation factor 4E, while New World arenaviruses do not, is only the second notable difference observed between New and Old World arenaviruses, the first being the difference in the glycoprotein receptor.

Project description:BackgroundDouble-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown.Principal findingsHuman heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection.SignificanceOur findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection.

Dataset Information

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junin Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Publications

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

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