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Host 5'-3' Exoribonuclease XRN1 Acts as a Proviral Factor for Measles Virus Replication by Downregulating the dsRNA-Activated Kinase PKR - PubMed

  • ️Sat Jan 01 2022

Host 5'-3' Exoribonuclease XRN1 Acts as a Proviral Factor for Measles Virus Replication by Downregulating the dsRNA-Activated Kinase PKR

Ethan BenDavid et al. J Virol. 2022.

Abstract

Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. IMPORTANCE Measles virus remains a major global health threat due to its high transmissibility and significant morbidity in children and immunocompromised individuals. Although there is an effective vaccine against MeV, a large population in the world remains without access to the vaccine, contributing to more than 7,000,000 measles cases and 60,000 measles deaths in 2020 (CDC). For negative-sense RNA viruses including MeV, one active research area is the exploration of virus-host interactions occurring at cytoplasmic IBs where viral replication takes place. In this study we present evidence suggesting a model in which MeV IBs antagonize host innate immunity by recruiting XRN1 to reduce dsRNA accumulation and subsequent PKR kinase activation/ISR induction. In the absence of XRN1, the increased dsRNA level acts as a potent activator of the antiviral PKR/ISR pathway leading to suppression of global cap-dependent mRNA translation and inhibition of viral replication.

Keywords: 5′-3′ exoribonuclease XRN1; inclusion body; integrated stress response; measles virus; protein kinase PKR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1

Impaired MeV replication in XRN1 knockout cells. Parental wild-type (WT) and XRN1 knockout (KO) A549 cells were infected with MeV using an MOI of 1.0. At 24 hpi one set of cells were lysed for viral protein analysis and a parallel set were used for virus yield analysis. (A) Infectious viral yields were determined and are plotted on a scatterplot. (B) Representative blots of MeV proteins are shown. (C) Quantification of individual MeV protein levels relative to the WT cells are presented as a bar chart. Both the scatterplot and bar chart represent the averages from 3 independent experiments. P values were calculated using Microsoft Excel Student's t test: *, P < 0.05; **, P < 0.001; ns, not significant.

FIG 2
FIG 2

Enhanced activation of the PKR kinase and ISR pathway in XRN1 knockout cells following MeV infection. WT and XRN1 KO A549 cells were left uninfected (as a control) or were infected, and processed as described in Fig. 1. (A) Lysates were analyzed by quantitative western immunoblotting to probe for PKR and ISR activation with representative blots shown. (B) The bar charts represent the average from 3 independent infected sample sets. P values were calculated using Microsoft Excel Student's t test: *, P < 0.05; **, P < 0.001; ns, not significant. (C) Positive control lysates were analyzed by quantitative western immunoblotting to verify the decreased gel mobility (indicated by arrows) of activated PERK after treatment with thapsigargin (TG) at 400 nM for 4 h, and activated GCN2 after serum starvation for 24 h (top). WT and XRN1 KO cell lysates from (A) were then probed for activation of PERK and GCN2 (bottom).

FIG 3
FIG 3

Increase of IFN-β induction and detectable RNase L activation in XRN1 knockout cells relative to parental wild-type cells following MeV infection. WT and XRN1 KO A549 cells were infected as described in Fig. 1. (A) and (C) At 24 hpi total RNA was extracted from uninfected and infected cells. (A) Total RNA from WT and XRN1 KO A549 cells were reverse transcribed into cDNA, and the relative IFN-β mRNA transcript levels determined using qPCR (top), as well as 5 different interferon-stimulated genes: MxA, MxB, ISG56, ISG54, and ISG60 (bottom). The bar charts represent the averages from 3 independent experiments. P values were calculated using Microsoft Excel Student's t test (top) or GraphPad Prism (version 9.2.0) one-way ANOVA and Šidák’s multiple-comparison test for selected pairs (bottom): *, P < 0.05; **, P < 0.001; ns, not significant. (B) At 24 hpi a parallel set of cells were lysed and the level of ISG15 was quantified using western immunoblotting. (C) Total RNA from WT and XRN1 KO A549 cells was analyzed using a Bioanalyzer on an RNA pico chip, including WT and RNase L KO A549 cells transfected with 1 μg/mL poly(I·C) for 6 h as a positive control for 28s/18s rRNA cleavage (indicated by arrows).

FIG 4
FIG 4

Significant restoration of MeV replication in XRN1/PKR/RNase L triple knockout cells compared to XRN1 knockout cells. (A) to (C) WT, XRN1 KO, PKR/RNase L double KO (DKO), and XRN1/PKR/RNase L triple KO (TKO) A549 cells were infected and analyzed as described in Fig. 1. (A) Representative blots of MeV proteins. (B) Bar charts showing the relative average levels of F0 and F1 proteins produced in the different cell lines as determined from 3 independent experiments. (C) Infectious viral yields were determined and are plotted as a scatterplot. Each graph represents the average from 3 independent experiments. P values were calculated using Microsoft Excel Student's t test: *, P < 0.05; **, P < 0.001; ns, not significant. (D) WT and XRN1 KO A549 cells were transfected with a non-targeting control or siRNA targeting either RNase L or PKR on days 1, 3, and 5. Transfected cells were infected on day 7, lysed on day 8, and analyzed as described in Fig. 1. Representative blots are shown.

FIG 5
FIG 5

Enhanced MeV protein synthesis in XRN1 knockout cells after treatment with an ISRIB inhibitor. (A) Lysates from Fig. 4D were probed for eIF2α phosphorylation using western immunoblotting. Representative blots are shown. (B) and (C) WT and XRN1 KO A549 cells were seeded at comparable confluence and pretreated for 12 h with either 1 μM ISRIB inhibitor or an equal volume of DMSO solvent as a control. Cells were then infected and processed as described in Fig. 1. The ISRIB treatment continued throughout the 24 h of infection. (B) Representative blots of selected MeV protein levels are shown. (C) Quantification of selected MeV protein levels relative to the DMSO-treated WT cells is presented as a bar chart, representing the average from 3 independent experiments. P values were calculated using Microsoft Excel Student's t test: *, P < 0.05; **, P < 0.001; ns, not significant.

FIG 6
FIG 6

Disruption of processing bodies does not affect MeV replication. WT A549 cells were transfected with a non-targeting control or each of the 2 independent eIF4E-T siRNAs on days 1, 3, and 5. (A) Transfected cell were fixed and stained using antibodies specific to eIF4E-T (to verify the knockdown efficiency) and DCP1A (to label the processing bodies). (B) Transfected cells were infected on day 7, lysed on day 8, and analyzed as described in Fig. 1. Representative blots for ISR activation and selected MeV protein levels are shown.

FIG 7
FIG 7

Induced translocation of EGFP-XRN1 to inclusion bodies following infection. (A) Validation of utilizing the transfected EGFP-XRN1 fusion for the live-cell imaging reporter as shown by its expected enrichment within the processing bodies (PBs) marked by DCP1A. (B) WT A549 cell lines stably expressing either EGFP or EGFP-XRN1 were infected with MeV expressing mCherry (containing a nuclear localization signal for reference of infected cells) using an MOI of 1.0. At 24 hpi live cells were imaged under a fluorescence microscope to capture representative images of EGFP-XRN1’s MeV-induced localization to large cytoplasmic puncta. (C) WT A549 cells stably expressing WDR5-mCherry fusion were transfected with either EGFP or EGFP-XRN1 plasmids, and then infected using an MOI of 1.0. At 24 hpi live transfected cells were imaged under a fluorescence microscope to capture representative images of EGFP-XRN1’s colocalization with viral IBs using WDR5-mCherry as a live marker for MeV IB.

FIG 8
FIG 8

Increased accumulation of dsRNA within MeV inclusion bodies in XRN1 knockout cells. (A) WT and XRN1 KO A549 cells were infected using an MOI of 0.1. At 36 hpi cells were fixed and stained using antibodies specific to dsRNA (J2) or MeV P (to label viral IBs), and then imaged under a fluorescence microscope to capture representative images (top). The bar chart shows the average percentage of infected cells with dsRNA enriched IBs from 3 independent experiments with 200 infected cells counted for each group per experiment (bottom). P values were calculated using Microsoft Excel Student's t test: *, P < 0.05; **, P < 0.001; ns, not significant. (B) WT and XRN1 KO A549 cells were infected using an MOI of 0.1. Cells were fixed and stained using the J2 and P antibodies and scored at different time intervals of infection (18, 20, 24, 30, and 36 hpi).

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References

    1. Lamb RA, Parks GD. 2013. Paramyxoviridae:the viruses and their replication, p 957–995. In Fields BN, Knipe DM, Howley PM (eds), Fields Virology: Sixth Edition. Lippincott, Williams, and Wilkins. Philadelphia, PA, USA.
    1. Brindley MA, Chaudhury S, Plemper RK. 2015. Measles virus glycoprotein complexes preassemble intracellularly and relax during transport to the cell surface in preparation for fusion. J Virol 89:1230–1241. 10.1128/JVI.02754-14. - DOI - PMC - PubMed
    1. Lin LT, Richardson CD. 2016. The host cell receptors for measles virus and their interaction with the viral hemagglutinin (H) protein. Viruses 8:250. 10.3390/v8090250. - DOI - PMC - PubMed
    1. Cattaneo R, Kaelin K, Baczko K, Billeter MA. 1989. Measles virus editing provides an additional cysteine-rich protein. Cell 56:759–764. 10.1016/0092-8674(89)90679-X. - DOI - PubMed
    1. Bellini WJ, Englund G, Rozenblatt S, Arnheiter H, Richardson CD. 1985. Measles virus P gene codes for two proteins. J Virol 53:908–919. 10.1128/JVI.53.3.908-919.1985. - DOI - PMC - PubMed

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