Hepatitis C virus infection is inhibited by a non-canonical antiviral signaling pathway targeted by NS3-NS4A

Vazquez, Christine; Tan, Chin Yee; Horner, Stacy M.

doi:10.1101/625640

Cited by 3 publications

(3 citation statements)

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“…reduced the relative m 6 A enrichment of CIRBP, consistent with that seen following infection of the parental cells (Figures S2E and 2C) (Vazquez et al, 2019). This suggests that IRF3 activation contributes to increased RIOK3 m 6 A modification while not affecting the m 6 A status of CIRBP.…”

Section: Resultssupporting

confidence: 80%

“…Flaviviridae infection drives signaling cascades that lead to the induction of interferon-b (IFN) and antiviral IFN-stimulated genes (ISGs) by IRF3 (Horner and Gale, 2013;Muñ oz-Jordá n and Fredericksen, 2010;Suthar et al, 2013). In infected Huh7 IRF3 KO cells (Vazquez et al, 2019), the increase in RIOK3 m 6 A with infection was attenuated (from $4to $1.5-fold) compared to parental cells (Figures S2E and 2C). However, DENV and ZIKV infection of IRF3 KO cells still S2 and S3.…”

Section: Resultsmentioning

confidence: 99%

“…Cell culture Huh7 and Huh-7.5 cells (gift of Dr. Michael Gale Jr., University of Washington (Sumpter et al, 2005)), Huh7 IRF3 KO cells (Vazquez et al, 2019), 293T cells (ATCC: CRL-3216) Vero cells (ATCC: CCL-81), C6/36 (ATCC: CRL-1660) were grown in Dulbecco's modification of Eagle's medium (DMEM; Mediatech) supplemented with 10% fetal bovine serum (HyClone), 25 mM HEPES (Thermo Fisher), and 1X non-essential amino acids (Thermo Fisher), referred to as complete DMEM (cDMEM). Huh7 and Huh-7.5 cells were verified using the Promega GenePrint STR kit (DNA Analysis Facility, Duke University), and cells were verified as mycoplasma free by the LookOut Mycoplasma PCR detection kit (Sigma-Aldrich).…”

Section: Experimental Model and Subject Detailsmentioning

confidence: 99%

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Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Experimental Model and Subject Detailsmentioning

confidence: 99%

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Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

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Signaling from the RNA sensor RIG-I is regulated by ufmylation

Snider

Park

Murphy

et al. 2021

Preprint

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The RNA binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFN). This signal transduction occurs at endoplasmic reticulum (ER)-mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, UFL1, as one of the proteins recruited to membranes at ER-mitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We find that following RNA virus infection, UFL1 is recruited to the membrane targeting protein 14-3-3ε, and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, loss of ufmylation prevents 14-3-3ε interaction with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a post-translational control for IFN induction.Significance StatementThe viral RNA sensor RIG-I initiates the antiviral innate immune response by activating a signaling cascade that induces interferon. Activation of the RIG-I signaling pathway is highly regulated to quickly mount a protective immune response while preventing dysregulation that can lead to excessive inflammation or autoimmune disorders. Here, we characterize one such mechanism of regulation. We describe that UFL1, an E3 ligase for the ubiquitin-like modifier conjugation system called ufmylation, is important to promote RIG-I signaling. Using molecular approaches, we show that ufmylation promotes RIG-I interaction with the membrane targeting protein 14-3-3ε. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor proteins MAVS for induction of interferon in response to RNA virus infection.

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Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

Vazquez

Tan

Horner

2019

J Virol

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The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that the addition of Riplet to these cells reduced HCV Y16F replication, whereas the addition of Riplet lacking the RING domain restored HCV Y16F replication. In addition, TBK1 inhibition or IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a noncanonical Riplet-TBK1-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection. IMPORTANCE The HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. Since Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.

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Hepatitis C virus infection is inhibited by a non-canonical antiviral signaling pathway targeted by NS3-NS4A

Cited by 3 publications

References 56 publications

Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

Signaling from the RNA sensor RIG-I is regulated by ufmylation

Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

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