Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection

  1. Sarah E Dremel
  2. Takanobu Tagawa
  3. Vishal N Koparde
  4. Carmen Hernandez-Perez
  5. Jesse H Arbuckle
  6. Thomas M Kristie
  7. Laurie T Krug
  8. Joseph M Ziegelbauer

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Abstract

To globally profile circRNAs, we employ RNA-Sequencing paired with chimeric junction analysis for alpha-, beta-, and gamma-herpesvirus infection. We find circRNAs are, as a population, resistant to host shutoff. We validate this observation using ectopic expression assays of human and murine herpesvirus endoribonucleases. During lytic infection, four circRNAs are commonly induced across all subfamilies of human herpesviruses, suggesting a shared mechanism of regulation. We test one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs are upregulated by either interferon-β or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we find an interferon-stimulated circRNA, circRELL1, inhibits lytic Herpes Simplex Virus-1 infection. We previously reported circRELL1 inhibits lytic Kaposi sarcoma-associated herpesvirus infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors characterized circRNAs that can escape degradation in cells infected by alpha-, beta and gamma herpesviruses both in cultured cells and in mice. During lytic infection, ten circRNAs were modulated across the virus subfamilies, and 67 circRNAs were upregulated after virus infection or treatment with interferon- or interferon-. The authors examined in detail interferon-induced circRNA circRELL1 and noted that this circRNA suppresses lytic infection, likely by interacting with the mTOR pathway and promoting cell proliferation. They also made the astonishing observation that the circRNAs were more resistant to cleavage by virus-encoded nucleases than their linear counterparts. This is a comprehensive study that reveals circRNA key players that control lytic and latent herpesvirus infections.

    Comments:

    1. Linear mRNA abundances are decreased after infection, but linear-derived circRNA abundances are upregulated. What determines increased circRNA abundances when their linear counterparts become limiting? Is the rate of splicing/back-splicing altered in infected cells? Is nuclear-cytoplasmic transport of circRNAs changed?
    2. CircRELL1 loss of function experiment: Does the employed siRNA affect the abundance of linear RELL1 mRNA?
    3. CircRELL1 gain of function experiment: How was this performed?
    4. Why are circRNA and, especially circRELL1, resistant to degradation in interferon-treated cells when OAS genes, and presumably RNAseL, are upregulated?

    Significance

    This study enhances our knowledge of circRNAs in viral infections.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Dremel et al explore the interplay between herpesvirus and circRNAs. This team has been a pioneer in the field and has made important discoveries about the regulation of circRNAs during herpesviral infection. While past work focused on the gamma-herpesviruses, here in this study, they expand their work to alpha and beta HV as well as extending their findings into animal models. They found consistent increase in CircRNA levels and found that these circRNAs are mostly resistant to viral-induced RNA decay. They then uncover that these circRNA can be induced by interferon which indicates that circRNAs could act as a first line of anti-viral defense. Consistently, they found that circRELL1, previously identified as a circRNA inhibiting KSHV lytic infection, can also restrict HSV-1. This seemingly conserved anti-viral capacity could point to an evolutionarily conserved mechanism of defense against infection. This study combines together an impressive amount of sequencing data and elegantly draws parallels between the various HV. While this is a complex story, bringing together expression levels of mRNA, circRNA and even diving into miRNA networks, this is extremely well written and as a reader, you feel transported into a well-crafted journey that keeps uncovering novel and exciting findings.

    Major comments:

    • there is a back and forth between the HVs that are included in the study: fig 1 has HSV, CMV and KSHV; while fig 2 is HSV, KSHV and MHV68. For clarity and consistency, the authors should consider including all 4 representative HV in their figures.
    • this might be a misunderstanding that just needs clarification: CIRCScores provide a measure of CircRNA reads over their linear counterparts: during host shutoff, the linear counterpart would likely decrease and therefore the CIRCscore would artificially go up. So the fact that the CIRCscore remains constant over lytic reactivation in KSHV and MHV68, wouldn't that indicate that instead the CircRNA level go down?
    • what happens to circRNAs in HCMV infection as they do not encode an endoRNase?
    • line 238: the authors should discuss why CpG and poly I:C treatment largely failed to induce expression of CircRELL1
    • line 289: if CircRELL1 is induced in response to infection, it could be interesting to induce its expression after infection (maybe a DOX-inducible promoter on the lentivirus) instead of prior to infection (which might hinder infection and hide more significant effects later).
    • line 329: the authors mention that the mRNA targets of SOX carry a "degenerate motif": do the circRNA downregulated during KSHV infection contain such motif?

    Minor comments:

    • figure 3B should show p-values
    • figure 3B: showing expression levels of the endoRNases could provide some context for the extent of their effect
    • I would refrain from using sentences referring to phenotype "trending upward" which basically reflects that the results are not significant in either upward or downward direction.

    Significance

    CircRNAs are only beginning to emerge as important regulators of gene expression in cells. Only recent developments in sequencing technology have allowed scientists to even detect the presence of these small RNA. Their roles and mechanism of induction remain largely uncharacterized, let alone in the context of viral infection. This team has pioneered the exploration of CircRNA in KSHV and is now poised to extend their findings to other members of the herpesvirus family. This study will be of interest to a broad audience, both virologist looking to better understand the viral-host battle during infection and RNA biologists seeking to better characterize how gene expression can be controlled with circRNA. There is also major therapeutic potential with these types of approaches.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Dremel et al reports on IFN-induced circRNAs that are produced in response to herpesviral infections and that are resistant to virus-mediated degradation (e.g. host shut-off). They provide a wealth of interesting data on circRNA production changes in response to infection with alpha, beta, and gamma herpesviruses and identify a number of host circRNAs that are commonly regulated across all subfamilies. Using circRELL1 as an example, they demonstrate a modest impact on productive HSV-1 infections (which follows up on their previous report of circRELL1 impacting KSHV lytic infection). They subsequently propose a new model in which a subset of IFN-stimulated genes produce both mRNAs and circRNAs with the potential for antiviral activity, the latter operating as a 'workaround' for avoiding virus-mediated shutoff.

    Major Comments

    • Line 146: The authors extended their analysis to HSV-1 latently infected mouse trigeminal ganglia but potentially missed a trick but not including an analysis of HSV-1/VZV infected human trigeminal ganglia for which potentially compatible datasets are available (PMID: 29563516).
    • Line 264: The authors note that a direct relationship in gene expression changes was observed between some circRNAs and mRNAs and not others. However, a gene level analysis may be problematic here as many genes encode multiple distinct transcript isoforms that are variably regulated e.g. during infection. A transcript level analysis (e.g. using Kallisto/Salmon) might enable the authors to specifically link circRNAs with individual mRNA isoforms which would be valuable information in the context of interferon-driven gene expression. Alternatively, the lack of a direct relationship in gene expression changes may also link to variability in circRNA decay / halflives. The authors could potentially solve this using a metabolic labelling approach to measure mRNA and circRNA decay rates for a subset of the genes of interest.
    • Line 273: The weak element in the paper relates to the role of circRELL1 in restricting HSV-1 productive infections. The effects observed are modest at best and the biological impact appears limited. It is also entirely unclear how circRELL1 might act to restrict HSV-1. The paper would significantly benefit from the authors extending their analysis to include 2-3 additional circRNAs that are commonly regulated by herpesviruses to determine (i) whether similar effects are observed and (iii) to determine whether a compound effect can be achieved by targeted silencing of multiple IFN-responsive circRNAs at the same time.

    Minor comments

    • Line 55: To this reviewers knowledge, only eight routinely infect humans (HSV1, HSV2, VZV, HCMV, HHV6, HHV7, EBV, and KSHV. It's possible the authors are considering HHV6A and HHV6B as distinct but this is not really the case.
    • Line 57: As written it implies that therapeutic agents capable of clearing VZV exist which is not really the case.
    • Line 132: The authors inclusion of previously published data (e.g. HCMV) along with reams of their own makes for a compelling analysis.
    • Line 582: A number of the sequencing datasets are not yet publicly available. This should be rectified before publication.

    Significance

    It is clear that a lot of work has gone into this manuscript and there a reams of data that will provide a great resource for mining to the wider herpesvirus community. While this naturally leads to a more descriptive nature, it does not undermine the value of the data. However, as indicated below, more functional validation is required if this work is to provide a significant step forward in our understanding of how and why IFN-induced circRNAs might be important for combating viral infections.

  5. Version published to 10.1038/s44319-023-00051-z
  6. Version published to 10.1101/2023.09.07.556698v1 on bioRxiv