Recognition of copy-back defective interfering rabies virus genomes by RIG-I triggers the antiviral response against vaccine strains

  1. Wahiba Aouadi
  2. Valérie Najburg
  3. Rachel Legendre
  4. Hugo Varet
  5. Lauriane Kergoat
  6. Frédéric Tangy
  7. Florence Larrous
  8. Anastassia V. Komarova
  9. Hervé Bourhy

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Abstract

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year around the world. A typical feature of RABV infection is the suppression of type I and III interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) to initiate IFN signaling remain elusive. Here, we showed that RABV RNAs are recognized by RIG-I (retinoic acid-inducible gene I) sensor resulting in an IFN response of the infected cells but that this global feature was differently modulated according to the type of RABV used. RNAs from pathogenic RABV strain, THA, were poorly detected in the cytosol by RIG-I and therefore mediated a weak antiviral response. On the opposite, we revealed a strong interferon activity triggered by the RNAs of the attenuated RABV vaccine SAD strain mediated by RIG-I. Using next-generation sequencing (NGS) combined with bioinformatics tools, we characterized two major 5’copy-back defective interfering (5’cb DI) genomes generated during SAD replication. Furthermore, we identified a specific interaction of 5’cb DI genomes and RIG-I that correlated with a high stimulation of the type I IFN signaling. This study indicates that RNAs from a wild-type RABV poorly activate the RIG-I pathway, while the presence of 5’cb DIs in vaccine SAD strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection and therefore strongly stimulates the IFN response.

Highlights

  • RABV pathogenic strain replication in vitro is characterized by the absence of defective interfering genomes thus induces a weak RLR-mediated innate immunity antiviral response.

  • RABV vaccine attenuated strain shows a high release of 5’ copy-back defective interfering genomes during replication in vitro and therefore enhances a strong antiviral response upon infection.

  • RIG-I is the main sensor for RABV RNA detection within cells.

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  1. Review Commons

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    Reply to the reviewers

    **Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.

    Reply to the reviewers

    **General Statements [optional]**

    Please find bellow the preliminary revision plan for our manuscript entitled “Recognition of copyback defective interfering rabies virus genomes by RIG-I triggers the antiviral response against vaccine strains” by Wahiba Aouadi et al. (RC-2022-01386). Reviewer’s’ comments/questions and suggestions are represented in blue in the text.

    **Description of the planned revisions**

    We thank the Reviewer#1 for underlining that identification of rabies virus 5’ copy-back DI genomes as “presumably bound to RIG-I is a useful advancement”, her/his interest in “observed difference between the responses to the two strains of virus” (THA strain and the vaccine SAD strain), and for emphasizing that “identification of the rabies viral RNAs that activate RIG-I is a significant finding for the rabies specialists”.

    Reviewer#1: In more details for the Evidence, reproducibility and clarity (Required) Much of the studies relied on weak methodologies. For example, in Fig 1, reporter assays were used, instead of measuring IFN mRNA levels; it is also not clear what is the nature of the promoter driving the reporter. Is it ISRE, which responds to IFN or is it the IFNb promoter, which responds to transcription factors activated by RIG-I? It is also not clear what is the nature of the RNA that was transfected. Is it total RNA from infected cells or is it purified viral RNA? No matter what, these results are quite predictable from the literature.

    Regarding the Reviewer #1 comment on type-I IFN cell report results “relying “on weak methodologies”, we would like to recall that to provide pieces of evidence that RIG-I-specific RNA ligands are produced during the infection with rabies virus we used several previously validated technics:

    • i) Fig.1A: transfected into ISRE-reporter cell line (ISRE, which responds to IFN) that is a classical validated tool to efficiently detect ISRE-activation even upon transfection of low quantities of immunoactive RNA ligands (PMID: 28768856, PMID: 27011352, PMID: 24098125, PMID: 29996094, PMID: 31761719, PMID: 23595062).
    • ii) Fig1B: Cell overexpressing LGP2 approach that has been previously developed and validated (Sanchez et al., 2019). LGP2 overexpressing cells provide a possibility to functionally distinguish between RIG-I and MDA5-driven activation of type-I IFN signaling. As noted in the corresponding figure legend in this experiment, the IFN-b promoter-reporter assay was used (“which responds to transcription factors activated by RIG-I”).
    • iii) Fig.1C -similar to Fig1A experiments performed in ISRE-reported cell lines partially depleted in either RIG-I or MDA5 (siRNA-based approach) to complement the Fig. 1B with additional functional validation using siRNAs.

    We apologize that we haven’t provided a detailed explanation for the origin of transfected RNA used all through Fig. 1. In the revised Fig1 we will correct the figure legend to explain the origin of total RNA used in experiments: Total RNA purified from SK.N.SH cells infected with THA or SAD for all experimental approaches presented in the figure. Moreover, as suggested by Reviewer#1 for Fig.1 we will add experiments measuring IFN-b mRNA by RT-qPCR.

    Referee #1

    Evidence, reproducibility and clarity

    A lot of effort was devoted to distinguish between RIG-I and MDA5 as the receptor of rabies viral RNA producing conflicting results from the binding assays and the reporter assays.

    This comment of Reviewer#1 is not clear to us. We have the feeling that our results do not show any conflict when analyzing the results represented in Fig. 2-3. They demonstrate that RIG-I and not MDA5 works as the key cytosolic sensor upon infection with rabies virus. Further, the apparent conflict observed by the Reviewer#1 about the fact that we failed to detect any specific RABV RNA ligands upon infection with THA strain (Fig.3A) while significant enrichment of immunoactive RNA ligands on RIG-I (Fig.2C) were observed can be easily commented and explained. We proposed in the revised version of our manuscript to discuss the possibility and to provide the results showing that enrichment in 5’PPP endogenous RNA ligands on RIG-I upon infection with THA RABV could explain the results observed on Fig.2C /Fig.3A. Indeed, in our recently accepted for publication study, we observed that a large spectrum of RNA virus infections leads to the mobilization of endogenous RNA ligands (transcripts of RNA Polymerase III) on RIG-I (https://www.cell.com/iscience/fulltext/S2589-0042(22)00871-9). Furthermore, we observed that upon infection Polymerase III transcripts can activate RIG-I signaling pathways even in the absence of RIG-I-specific viral RNA ligands. To address this possibility in the revised manuscript, we propose to perform additional analysis of our RNAseq results to demonstrate enrichment of endogenous RNA ligands on RIG-I in rabies virus-infected cells.

    Significance

    Conceptually, the paper does not add much to the literature. As pointed out by the authors, RIG-I-specific partners had been identified before for many RNA viruses including other rhabdoviruses.

    We additionally underline that although there is a slowly growing number of studies characterizing RLR-specific RNA ligands directly from infected cells with a slowly growing number of characterized viruses, to our knowledge our study provides the first characterization of RLRspecific RNA ligands in Rabies virus-infected cells and that the amount of these ligands differs between wild type viruses and vaccine strains. Furthermore, none of the previously published studies on Rabies virus used similar experimental approaches. We believe that only stepwise characterization of RLR-specific RNA ligands for different RNA virus families is fully original regarding rabies virus and will further provide a wider and more fundamental vision on the distribution of RIG-I and MDA5 specificities for sensing RNA viruses.

    Referee #3

    Evidence, reproducibility and clarity

    We thank Reviewer#3 for stressing that our “study is highly significant for understanding virus sensing mechanisms and to inform understanding of vaccine actions.” For the Reviewer#3 specific comments:

    The signaling analyses is focused on ISRE/promoter induction, which is several steps downstream from RIG-I. An more comprehensive signaling analysis is required to define the RLR pathway engagement, including examination of RIG-I binding to MAVS, IRF3 activation induced by viral RNA and recovered RIG-I or MDA5 ligands, and induction of IRF3-target gene expression (such as RSAD, IFI44, IFIT1, IFIT2) and interferon-stimulated gene (ISG) expression such as Mx1, Mx1, OAS, etc.

    We thank Reviewer#3 for his comments and also appreciate that additional characterization of type-I IFN signaling pathway activation by RABV RNA will deeper our research results. We will add additional experimental results to answer the comments suggested by the Reviewer#3 for each Figure, as presented below:

    Figure 1. RLR activation readout here relies exclusively on promoter/reporter assay. Assessment of endogenous IRF3, IRF3-target gene expression, and ISG expression needs to be included. Also, what are the dynamics of RLR signaling activation during infection over a time course? This is important to know and to associate with the accumulation of the cb RNAs.

    We will perform additional transfection of total RNA purified from SK.N.SH cells infected with THA or SAD to HEK293T (or other relevant cells) to detect by WB analysis the phosphorylation of IRF3. As suggested by the Reviewer#3 we will also perform gene expression analysis targeting RSAD, IFI44, IFIT1, IFIT2. Additionally, kinetics of the SK.N.SH cells infection with THA or SAD strains of RABV will be studied to detect the accumulation of 5’cbDI genomes during the infection as suggested at the second part of the comment by the Reviewer#3.

    Figure 2. The RLR-bound RNA signaling analysis is incomplete. The authors need to include analysis of IRF3 and gene expression as noted above. Also, the authors should assess RLRbound RNAs collected over a time course of infection, thus enabling an understanding of the temporal dynamics of RLR ligand and biological activity of this virus-host interaction.

    In order to reply to this comment we will provide additional characterization of type-I IFN signaling in ST-RLR cells infected with THA and SAD, comparing to the mock-infected cells. For this, we will perform western blot analysis of IRF3P in total protein lysates and carry additional analysis of our NGS data to visualize ISG expression profiles in the same conditions (THA, SAD, and mock). Unfortunately, it will be experimentally difficult to assess RLR-bound RNAs collected over a time course of infection. However, as our NGS analysis demonstrated accumulation of 5’cb DI RNA as specific RNA ligands of RIG-I, we can follow the kinetics of accumulation of these 5’cb DI RNAs in SK.N.SH and ST-RLR cells as described above in response to the Fig.1 comment of the Reviewer#3.

    Figure 3. These are strong data sets and are convincing. For panel C, one can see several RIGI-bound peaks. The authors should provide more information on the length of these peaks, please include in Table 1. Also for MDA5 there also are peaks but the histogram is saturated. The peaks and valleys need to be delineated, ideally in a large table. The needs to be confirmation of these motifs or RNAs as actually binding to RIG-I and MDA5. This binding activity needs to be shown in gel-shift assay or other suitable approach of direct RIG-I binding of specific RNAs produced in vitro corresponding to mapped regions shown in the figure 3. Also, a more careful analysis of MDA5-assocaited RNA needs to be conducted to ascertain if it has immune stimulatory/signaling activity. By assess IRF3 activation this activity might be identified.

    Based on the Reviewer#3 suggestions for the Fig.3C we will additionally summarize in Supplementary Table 4 RNA reads that are represented as enriched on RIG-I for the 5’ part of the RABV genome. Indeed, the full-length genome binding to MDA5 was observed for RNA- reads importantly in SAD-infected cells. However, we believe that how encapsidated full-length viral genome can still be detected by MDA5 in virus-infected cells needs to be addressed in a separate study. Additional experiments for detecting the IRF3 activation in ST-RLR cells will be performed as described above.

    Figure 4: VERY important: Do these RNAs bind to RIG-I in vitro, and do they activate IRF3 when transfected into cells, what is the role of 5'ppp in this activity?? These data are needed to make the strong conclusions stated by the authors.

    We are grateful to Reviewer#3 suggestions for Fig.4. We will address whether the detected RABV 5’cb DI RNAs are specific RIG-I ligands. We will synthetize and transfect these RNA molecules and study how efficiently they activate type-I IFN signaling (by IFN-b and ISRE reporter approaches as well as by gene expression assay analysis as suggested in Fig.1 by Reviewer#3). We will also address IRF3P efficiency upon cell transfection with DI-2170 and DI-1668. As controls, we will use previously described RIG-I/MDA5-specific RNA ligands and treat RNA transcripts with calf intestine alkaline phosphatase (CIP) to remove 5’ppp groups.

    **Description of the revisions that have already been incorporated in the transferred manuscript**

    No revisions have already been incorporated in the transferred manuscript.

    **Description of analyses that authors prefer not to carry out**

    As described above to answer to the Reviewer#3 suggestion, how encapsidated full-length viral genome can still be detected by MDA5 in virus-infected cells needs to be addressed in a separate study.

    Referee #2

    Evidence, reproducibility and clarity

    We thank the Reviewer#2 for underlining that our study “shed light on the RLR recognition of RABV RNAs upon infection” and that our study “clarify the mechanism of cellular immunity differences between RABV pathogenic strain and vaccine attenuated strain. Reviewer#2 suggested to “verify whether the difference in this mechanism is caused by the difference in the viral genome, whether the N gene or L gene of the two can be exchanged by reverse genetics, and then infect the cells to verify whether the 5'cb DI genomes can be generated just as this paper.”

    We agree with Reviewer#2 that applying reverse genetics for RABV genome by exchanging N and L genes could provide a more in-depth characterization of 5’cb DI generation and pathogenicity of RABV. However, these additional experiments cannot be provided within the scope of this paper and will take time for the revision process. We believe, that this question needs to be addressed in a separate study by exchanging either N and L genes using reverse genetics.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Bourhy and colleagues present their study focused on defining how rabies virus (RV) vaccine strains trigger RIG-I innate immune signaling. Triggering RIG-I or MDA5 leads to innate immune activation, and in vaccinology this is an important component of immune adjuvant actions that serve to overall enhance vaccine immunity. The group applied in vitro infection and RNA analyses including assessment of RLR-dependent signaling by RNAs recovered from infected cells, and RNAseq of RLR-associated RNA from virus-infected cells, showing that RIG-I binds to copyback (cb) RNAs of defective interfering (DI) genomes produced during replication by the RV vaccine strain SAD but not by THA strain which likely does not produce the cb RNAs. The study extends previous work showing that RIG-I senses RV RNA to now show That RIG-I binds to the cb RNAs. Data on MDA5 is included to show that MDa5 is bound across the RV negative strand but the RNA recovered from MDA5 in infected cells does not stimulate innate immune signaling.

    Specific comments:

    The signaling analyses is focused on ISRE/promoter induction, which is several steps downstream from RIG-I. An more comprehensive signaling analysis is required to define the RLR pathway engagement, including examination of RIG-I binding to MAVS, IRF3 activation induced by viral RNA and recovered RIG-I or MDA5 ligands, and induction of IRF3-target gene expression (such as RSAD, IFI44, IFIT1, IFIT2) and interferon-stimulated gene (ISG) expression such as Mx1, Mx1, OAS, etc.

    Figure 1. RLR activation readout here relies exclusively on promoter/reporter assay. Assessment of endogenous IRF3 , IRF3-target gene expression, and ISG expression needs to be included. Also what are the dynamics of RLR signaling activation during infection over a time course? This is important to know and to associate with the accumulation of the cb RNAs.

    Figure 2. The RLR-bound RNA signaling analysis is incomplete. The authors need to include analysis of IRF3 and gene expression as noted above. Also, The authors should assess RLR-bound RNAs collected over a time course of infection, thus enabling an understanding of the temporal dynamics of RLR ligand and biological activity of this virus-host interaction.

    Figure 3. These are strong data sets and are convincing. For panel C, one can see several RIG-I-bound peaks. The authors should provide more information on the length of these peaks, please include in Table 1. Also for MDA5 there also are peaks but the histogram is saturated. The peaks and valleys need to be delineated, ideally in a large table. The needs to be confirmation of these motifs or RNAs as actually binding to RIG-I and MDA5. This binding activity needs to be shown in gel-shift assay or other suitable approach of direct RIG-I binding of specific RNAs produced in vitro corresponding to mapped regions shown in the figure 3. Also, a more careful analysis of MDA5-assocaited RNA needs to be conducted to ascertain if it has immune stimulatory/signaling activity. By assess IRF3 activation this activity might be identified.

    Figure 4: VERY important: Do these RNAs bind to RIG-I in vitro, and do they activate IRF3 when transfected into cells, what is role of 5'ppp in this activity?? These data are needed to make the strong conclusions stated by the authors.

    Review cross-commenting:

    I agree completely with the comments provided by other two reviewers.

    Significance

    The study is highly significant for understanding virus sensing mechanisms and to inform understanding of vaccine actions.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript entitled by"Recognition of copy-back defective interfering rabies virus genomes by RIGI triggers the antiviral response against vaccine strains"，which clarify the mechanism of cellular immunity differences between RABV pathogenic strain and vaccine attenuated strain in vitro. This paper using next-generation sequencing (NGS) combined with bioinformatics tools, it was found that the RABV vaccine attenuated strain replication in vitro induces a high release of 5' copy-back defective interfering genomes, which enhances a strong antiviral response. However, RABV pathogenic strain replication in vitro is characterized by the absence of defective interfering genomes thus induces a weak RLR-mediated innate immunity antiviral response. This paper demonstrated that IFN response induced by RLR RABV RNA recognition was principally mediated by RIG-I. 5'cb DI viral genomes that enhance RIG-I detection and therefore strongly stimulate the IFN response were exclusively produced by the RABV vaccine strain. To verify whether the difference in this mechanism is caused by the difference in the viral genome, whether the N gene or L gene of the two can be exchanged by reverse genetics, and then infect the cells to verify whether the 5'cb DI genomes can be generated just as this paper.

    Review cross-commenting:

    I agree completely with the comments provided by other two reviewers.

    Significance

    This paper shed light on the RLR recognition of RABV RNAs upon infection.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Rabies virus, a rhabdovirus, is a major human pathogen and mammalian cells respond to infection by this virus by triggering type I IFN synthesis. Here, the authors report that the cytoplasmic antiviral sensor RIG-I recognizes viral RNA to initiate signaling. Moreover, the vaccine strain SAD activates RIG-I more effectively than the pathogenic strain, THA. During SAD replication, two major 5' copy-back defective interfering genomes were generated and they bound to RIG-I to activate it.

    Much of the studies relied on weak methodologies. For example, in Fig 1, reporter assays were used, instead of measuring IFN mRNA levels; it is also not clear what is the nature of the promoter driving the reporter. Is it ISRE, which responds to IFN or is it the IFNb promoter, which responds to transcription factors activated by RIG-I? It is also not clear what is the nature of the RNA that was transfected. Is it total RNA from infected cells or is it purified viral RNA? No matter what, these results are quite predictable from the literature. A lot of effort was devoted to distinguish between RIG-I and MDA5 as the receptor of rabies viral RNA producing conflicting results from the binding assays and the reporter assays. A major weakness of these experiments is in the use of convoluted cell lines which added to the weakness of the reporter assays as outlined above. Identification of the DI viral sequences that presumably bound to RIG-I is a useful advancement.

    Significance

    Conceptually, the paper does not add much to the literature. As pointed out by the authors, RIG-I-specific partners had been identified before for many RNA viruses including other rhabdoviruses. The observed difference between the responses to the two strains of virus is interesting but multiple strains need to be tested to make a meaningful interpretation of the data. Nonetheless, identification of the rabies viral RNAs that activate RIG-I is a significant finding for the rabies specialists.

    This reviewer's expertise is in antiviral innate immune response and the type I IFN system.

  5. Version published to 10.1101/2022.03.24.485606v1 on bioRxiv