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. 2008 Apr 30;3(4):e2032.
doi: 10.1371/journal.pone.0002032.

Processing of genome 5' termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction

Matthias Habjan  1 , Ida Andersson, Jonas Klingström, Michael Schümann, Arnold Martin, Petra Zimmermann, Valentina Wagner, Andreas Pichlmair, Urs Schneider, Elke Mühlberger, Ali Mirazimi, Friedemann Weber
Affiliations

Processing of genome 5' termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction

Matthias Habjan et al. PLoS One. .

Abstract

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5' triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5' triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5' monophosphate-specific exonuclease indicates that the RIG-I-activating 5' triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5' end as a strategy of viruses to evade the innate immune response.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genomic RNAs of ZEBOV and NiV activate the IFN response similar to FLUAV RNA.
Human 293T cells were transfected with luciferase reporter plasmids to measure activation of the inducible IFN-β promoter and the constitutively active SV40 promoter, respectively. At 6 h post-transfection, cells were either mock treated or transfected with 1 µg viral genomic RNA (vRNA) of ZEBOV (A), NiV (B), or FLUAV (C). After overnight incubation, cells were lysed and promoter activities were normalised to the mock-induced samples. Mean values and standard deviations from 3 independent experiments are shown. (D) Detection of mRNAs for IFN-β (panel 1) and the IFN-stimulated genes IP-10, ISG56, and OAS (panels 2 to 4). Detection of γ-actin mRNA served as control (panel 5). 293T cells were transfected with 1 µg vRNA or 5 µg of the dsRNA analog poly(IC) and monitored 18 h later for gene upregulation by RT-PCR analysis.
Figure 2
Figure 2. IFN induction by NSV vRNAs depends on RIG-I and the 5′ triphosphate group.
(A) Verification of knockdowns. Human 293T cells were treated with retroviral shRNA constructs directed against either RIG-I or MDA5, and cotransfected with expression constructs for HA-tagged MDA5 (left panels) or GFP-fused RIG-I (right panels). Western blot analysis using antibodies against the respective fusion tags is shown. Detection of cellular β-tubulin was used as an internal control. (B) Effect of shRNA knockdowns on IFN induction by viral RNAs, using the reporter constructs and RNA transfection protocols as described for Fig. 1A. The negative control shRNA construct (CTRL) targets the heat shock 70 interacting protein and was tested to have no effect on IFN induction (data not shown). (C) Genomic RNAs from ZEBOV and NiV were either mock treated, treated with SAP, or treated with SAP in the presence of the phosphatase inhibitor EDTA. IFN-β reporter assays and RNA transfections were performed as described for Fig. 1A. Mean values and standard deviations from 3 independent experiments are shown.
Figure 3
Figure 3. Genomic RNAs of segmented NSVs.
RNAs isolated from virus particles of LASV (A) and RVFV (B) were tested for their ability to activate the IFN-β promoter in dependency of RIG-I or MDA5 (left panels) or 5′ triphosphate groups (right panels). (C) IFN-β promoter activation by genomic RNAs of the bunyaviruses RVFV, HTNV, and CCHFV. Mean values and standard deviations from 3 independent experiments are shown. (D) Purified virion RNAs (5 µg) of RVFV, HTNV, and CCHFV were separated on denaturing formaldehyde agarose gels. The genome segments (L, large; M, middle; S, small) are labeled on the right, and asterisks indicate 28S and 18S rRNA bands. Contamination of virus preparations with ribosomal RNAs are often observed but have no consequences for IFN induction. As a control RNA isolated from ultracentrifuged supernatants of uninfected cells is shown (mock).
Figure 4
Figure 4. Non-inducing viral RNAs contain a 5′ monophosphate.
(A) vRNAs of RVFV (panel 1), HTNV (panel 2) and CCHFV (panel 3) were incubated with a 5′monophosphate-specific 5′-3′ exonuclease. After 4 h of incubation, digestion efficacy was tested by RT-PCR analysis using primer pairs specific for the viral S segment. A comparison with untreated vRNAs is shown (lane input RNA). As additional controls, RNA was incubated without enzyme (lane buffer) or H2O was used for RT-PCR. The faint residual RT-PCR bands obtained after digestion of HTNV or CCHFV vRNAs are most likely caused by a minority of RNAs containing exonuclease-resistant 5′-OH ends. Such 5′-OH ends were previously observed for HTNV vRNA and thought to represent a preparation artifact . (B) Activation of the IFN-β promoter by genomic RNAs isolated from MV and BDV particles. Mean values and standard deviations from 3 independent experiments are shown. (C) Treatment of MV and BDV genomic RNAs with a 5′ monophosphate-specific 5′-3′ exonuclease and subsequent RT-PCR analysis.
Figure 5
Figure 5. vRNA binding by RIG-I.
GFP-RIG-I expressed by 293T cells was coupled to protein G Sepharose beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H2O was used as negative control.
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