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hnRNPA2B1: Fueling Antiviral Immunity from the Nucleus
Molecular Cell
Spotlight hnRNPA2B1: Fueling Antiviral Immunity from the Nucleus Fiachra Humphries1 and Katherine A. Fitzgerald1,* 1University of Massachusetts Medical School, Worcester, MA 01655, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2019.09.021
In a recently published article in Science, Cao and colleagues (Wang et al., 2019) identify hnRNPA2B1 as a new DNA-binding protein that initiates and amplifies antiviral immunity, unveiling a new facet of DNA recognition in the nucleus. Detection of microbial dsDNA and induction of type I interferon (IFN) responses is a central feature of antiviral immunity. Cyclic GMP-AMP synthase (cGAS) is a key dsDNA sensor important in controlling virus infection. When cGAS binds dsDNA, cGAMP is generated that then binds STING to activate autophagy or TBK1IRF3 and IKK-NF-kB signaling resulting, respectively, in rapid transcription of type I IFNs and proinflammatory cytokines (Liu et al., 2019). Herpesviruses such as HSV1, however, primarily infect epithelial cells and deliver their genomic dsDNA material directly to the nucleus of infected cells (Enquist and Leib, 2016). While other DNA sensors of HSV-1 such as IFI16 are localized in the nucleus, it has been unclear how and where cGAS ligation by DNA occurs during infection with this class of virus. Very recent work positions cGAS in the nucleus of some cells, providing some clues to these processes (Zierhut et al., 2019). In this new study, Cao and coworkers identify hnRNPA2B1 as a nuclear receptor for viral DNA critical for the host responses to herpesvirus infection. They demonstrate that hnRNPA2B1 initiates a cGAS-independent pathway via TBK1IRF3 to turn on type I IFNs. In addition, they define a second role for this protein, where hnRNPA2B1 also controls the export of cGAS and STING mRNAs that in turn amplify antiviral responses (Figure 1) (Wang et al., 2019). Using a proteomics approach, Cao and colleagues searched for factors that bound viral dsDNA and translocated from the nucleus to the cytoplasm during HSV-1 infection. siRNA knockdown validation of candidates led to the identification of hnRNPA2B1 as a new regulator of HSV-1 infection. hnRNPA2B1 is a
hnRNP family member. hnRNPs play important roles in RNA biology and the regulation of transcriptional responses. Indeed, hRNPA2B1 associates with lincRNA-Cox2 to negatively regulate TLR-induced cytokine responses (Carpenter et al., 2013). In this report, the authors found that knocking down hnRNPA2B1 in macrophages attenuated the IFNb response during HSV-1 infection. They also found that hnRNPA2B1 binds viral DNA, and this is tightly controlled by post-translational modifications (PTMs). In resting cells, hnRNPA2B1 is methylated while in virus-infected cells, hnRNPA2B1 is demethylated by JMJD6. Demethylated hnRNPA2B1 then undergoes dimerization and exits the nucleus. The accumulated hnRNPA2B1 in the cytosol associates with and activates TBK1, enhancing IRF3 phosphorylation and transcription of IFNb. The physiological relevance of hnRNPA2B1 was determined through generation of hnRNPA2B1 myeloid cell conditional knockout (cKO) mice. hnRNPA2B1 cKO mice were highly susceptible to HSV-1 infection due to loss of the type I IFN response. Indeed, hnRNPA2B1 cKO mice exhibited significant increases in mortality and viral titers following intraperitoneal HSV-1 infection compared to wildtype mice. Importantly, hnRNPA2B1 only impacted the TBK1-IRF3 pathway controlling the IFN response, as NF-kB responses were unaffected in cells lacking hnRNPA2B1. Multiple lines of evidence, including robust in vivo data, have highlighted the central role of the cGASSTING pathway as a driver of TBK1-IRF3 activation and type I IFN production during HSV-1 infection. Exactly how cGAS and hnRNPA2B1 function in time and
8 Molecular Cell 76, October 3, 2019 ª 2019 Elsevier Inc.
space to initiate this signaling pathway is an important point to consider further. The Cao study adds some insight to this by demonstrating that hnRNPA2B1 also functions as an amplifier of the cGASSTING pathway. This occurs through m6A modifications on cGAS and STING mRNAs, which facilitate their export to the cytosol to be translated. This function of hnRNPA2B1 then boosts the cGAS-STING pathway. cGAS-STING-mediated autophagy is also an important mechanism restricting viral replication, and uncovering any cross-talk between hnRNPA2B1 and autophagy may further validate the importance of hnRNPA2B1 during HSV-1 infection. The studies by Cao and colleagues using cKO mice are restricted to macrophages, as mice lacking hnRNPA2B1 more broadly in other cell types were not examined. Herpesviruses replicate in epithelial cells, and studies of hnRNPA2B1 function in restricting the virus in this setting would be informative (Enquist and Leib, 2016). The natural route of HSV-1 infection occurs through retrograde transport from the eye to the CNS, where the cGAS-STING pathway is activated following phagocytosis of infected neurons by microglia to control neuroinvasive strains of HSV-1 (Reinert et al., 2016). cGAS and STING are indispensable in controlling the virus in this setting. Thus, it will be of interest to determine how hnRNPA2B1 functions in more physiologically relevant routes of infection. Comparisons of hnRNPA2B1 to cGAS are also instantly drawn by the ability of both proteins to recognize not only pathogen-derived but also host DNA. Failure to control DNA accumulation drives a range of debilitating human diseases,
Molecular Cell
Spotlight
Figure 1. hnRNPA2B1 Amplifies the cGAS-STING Pathway during DNA Virus Infection (A) Demethylation, dimerization, and translocation of hnRNPA2B1 following detection of viral DNA. (B) Association and activation of TBK1 by hnRNPA2B1. (C) hnRNPA2B1 mediated m6A of cGAS, STING, and IFI16 mRNA.
such as Aicardi-Goutie`res syndrome (AGS), resulting from inactivating mutations in nucleases such as TREX1. Indeed, cGAS plays a critical role in recognizing the accumulated DNA, driving the pathophysiology of this condition (Gao et al., 2015; Mackenzie et al., 2017). Thus, it will be interesting to determine if hnRNPA2B1 sensing of host DNA also contributes to the pathophysiology of AGS and related conditions. In support of this, it is intriguing that autoantibodies to hnRNPA2B1 have been detected in patients with lupus (Dumortier et al., 2000) This study also expands on the importance of PTMs in the positive and negative regulation of DNA sensors. Methylation sequesters monomeric hnRNPA2B1 in the nucleus, whereas acetylation can control activity and cellular localization of cGAS and IFI16. Indeed, nuclear localization, cytoplasmic translocation, and epigenetic modifications are characteristics encompassing multiple DNA sensors including cGAS, IFI16/p204, and now hnRNPA2B1 (Dai et al., 2019; Li et al., 2012).
These findings expand the spatial regulation of DNA sensors to include sensors in the endosome (TLR9), cytosol (cGAS/ AIM2), and now the nucleus (hnRNPA2B1 and cGAS). The work also highlights the temporal regulation of antiviral pathways with distinct sensors affecting the priming and amplification of these pathways. cGAS, IFI16, and now hnRNPA2B1 form a triad of nuclear DNA sensors that collectively synergize to amplify STING activation and restrict HSV-1 replication. Future work investigating cross-talk between these receptors and their role in different cell types may uncover further layers of regulation of antiviral immunity. Lastly, this study also raises questions regarding the juxtaposition of nuclear and cytoplasmic DNA sensing and highlights how transfer of secondary signals across the nuclear membrane is an essential component of DNA sensing. Indeed, if cGAS is truly a nuclear protein, demonstrating nucleocytoplasmic cGAMP trafficking will be important. The study from Cao and colleagues illuminates new complexity to
DNA sensing and sheds new light on the communication between nuclear sensors and cytoplasmic effectors. ACKNOWLEDGMENTS This work is supported by grants from the NIH (AI128358 and AI067497).
REFERENCES Carpenter, S., Aiello, D., Atianand, M.K., Ricci, E.P., Gandhi, P., Hall, L.L., Byron, M., Monks, B., Henry-Bezy, M., Lawrence, J.B., et al. (2013). A long noncoding RNA mediates both activation and repression of immune response genes. Science 341, 789–792. Dai, J., Huang, Y.-J., He, X., Zhao, M., Wang, X., Liu, Z.-S., Xue, W., Cai, H., Zhan, X.-Y., Huang, S.-Y., et al. (2019). Acetylation Blocks cGAS Activity and Inhibits Self-DNA-Induced Autoimmunity. Cell 176, 1447–1460.e14. Dumortier, H., Monneaux, F., Jahn-Schmid, B., Briand, J.-P., Skriner, K., Cohen, P.L., Smolen, J.S., Steiner, G., and Muller, S. (2000). B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice. J. Immunol. 165, 2297–2305.
Molecular Cell 76, October 3, 2019 9
Molecular Cell
Spotlight Enquist, L.W., and Leib, D.A. (2016). Intrinsic and Innate Defenses of Neurons: De´tente with the Herpesviruses. J. Virol. 91, e01200–e01216. Gao, D., Li, T., Li, X.-D., Chen, X., Li, Q.-Z., WightCarter, M., and Chen, Z.J. (2015). Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. USA 112, E5699–E5705. Li, T., Diner, B.A., Chen, J., and Cristea, I.M. (2012). Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc. Natl. Acad. Sci. USA 109, 10558–10563.
10 Molecular Cell 76, October 3, 2019
Liu, D., Wu, H., Wang, C., Li, Y., Tian, H., Siraj, S., Sehgal, S.A., Wang, X., Wang, J., Shang, Y., et al. (2019). STING directly activates autophagy to tune the innate immune response. Cell Death Differ. 26, 1735–1749. Mackenzie, K.J., Carroll, P., Martin, C.-A., Murina, O., Fluteau, A., Simpson, D.J., Olova, N., Sutcliffe, H., Rainger, J.K., Leitch, A., et al. (2017). cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465. Reinert, L.S., Lopusna´, K., Winther, H., Sun, C., Thomsen, M.K., Nandakumar, R., Mogensen, T.H., Meyer, M., Vægter, C., Nyengaard, J.R.,
et al. (2016). Sensing of HSV-1 by the cGASSTING pathway in microglia orchestrates antiviral defence in the CNS. Nat. Commun. 7, 13348. Wang, L., Wen, M., and Cao, X. (2019). Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science 365, eaav0758. Zierhut, C., Yamaguchi, N., Paredes, M., Luo, J.D., Carroll, T., and Funabiki, H. (2019). The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death. Cell 178, 302–315.e23.
Spotlight hnRNPA2B1: Fueling Antiviral Immunity from the Nucleus Fiachra Humphries1 and Katherine A. Fitzgerald1,* 1University of Massachusetts Medical School, Worcester, MA 01655, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2019.09.021
In a recently published article in Science, Cao and colleagues (Wang et al., 2019) identify hnRNPA2B1 as a new DNA-binding protein that initiates and amplifies antiviral immunity, unveiling a new facet of DNA recognition in the nucleus. Detection of microbial dsDNA and induction of type I interferon (IFN) responses is a central feature of antiviral immunity. Cyclic GMP-AMP synthase (cGAS) is a key dsDNA sensor important in controlling virus infection. When cGAS binds dsDNA, cGAMP is generated that then binds STING to activate autophagy or TBK1IRF3 and IKK-NF-kB signaling resulting, respectively, in rapid transcription of type I IFNs and proinflammatory cytokines (Liu et al., 2019). Herpesviruses such as HSV1, however, primarily infect epithelial cells and deliver their genomic dsDNA material directly to the nucleus of infected cells (Enquist and Leib, 2016). While other DNA sensors of HSV-1 such as IFI16 are localized in the nucleus, it has been unclear how and where cGAS ligation by DNA occurs during infection with this class of virus. Very recent work positions cGAS in the nucleus of some cells, providing some clues to these processes (Zierhut et al., 2019). In this new study, Cao and coworkers identify hnRNPA2B1 as a nuclear receptor for viral DNA critical for the host responses to herpesvirus infection. They demonstrate that hnRNPA2B1 initiates a cGAS-independent pathway via TBK1IRF3 to turn on type I IFNs. In addition, they define a second role for this protein, where hnRNPA2B1 also controls the export of cGAS and STING mRNAs that in turn amplify antiviral responses (Figure 1) (Wang et al., 2019). Using a proteomics approach, Cao and colleagues searched for factors that bound viral dsDNA and translocated from the nucleus to the cytoplasm during HSV-1 infection. siRNA knockdown validation of candidates led to the identification of hnRNPA2B1 as a new regulator of HSV-1 infection. hnRNPA2B1 is a
hnRNP family member. hnRNPs play important roles in RNA biology and the regulation of transcriptional responses. Indeed, hRNPA2B1 associates with lincRNA-Cox2 to negatively regulate TLR-induced cytokine responses (Carpenter et al., 2013). In this report, the authors found that knocking down hnRNPA2B1 in macrophages attenuated the IFNb response during HSV-1 infection. They also found that hnRNPA2B1 binds viral DNA, and this is tightly controlled by post-translational modifications (PTMs). In resting cells, hnRNPA2B1 is methylated while in virus-infected cells, hnRNPA2B1 is demethylated by JMJD6. Demethylated hnRNPA2B1 then undergoes dimerization and exits the nucleus. The accumulated hnRNPA2B1 in the cytosol associates with and activates TBK1, enhancing IRF3 phosphorylation and transcription of IFNb. The physiological relevance of hnRNPA2B1 was determined through generation of hnRNPA2B1 myeloid cell conditional knockout (cKO) mice. hnRNPA2B1 cKO mice were highly susceptible to HSV-1 infection due to loss of the type I IFN response. Indeed, hnRNPA2B1 cKO mice exhibited significant increases in mortality and viral titers following intraperitoneal HSV-1 infection compared to wildtype mice. Importantly, hnRNPA2B1 only impacted the TBK1-IRF3 pathway controlling the IFN response, as NF-kB responses were unaffected in cells lacking hnRNPA2B1. Multiple lines of evidence, including robust in vivo data, have highlighted the central role of the cGASSTING pathway as a driver of TBK1-IRF3 activation and type I IFN production during HSV-1 infection. Exactly how cGAS and hnRNPA2B1 function in time and
8 Molecular Cell 76, October 3, 2019 ª 2019 Elsevier Inc.
space to initiate this signaling pathway is an important point to consider further. The Cao study adds some insight to this by demonstrating that hnRNPA2B1 also functions as an amplifier of the cGASSTING pathway. This occurs through m6A modifications on cGAS and STING mRNAs, which facilitate their export to the cytosol to be translated. This function of hnRNPA2B1 then boosts the cGAS-STING pathway. cGAS-STING-mediated autophagy is also an important mechanism restricting viral replication, and uncovering any cross-talk between hnRNPA2B1 and autophagy may further validate the importance of hnRNPA2B1 during HSV-1 infection. The studies by Cao and colleagues using cKO mice are restricted to macrophages, as mice lacking hnRNPA2B1 more broadly in other cell types were not examined. Herpesviruses replicate in epithelial cells, and studies of hnRNPA2B1 function in restricting the virus in this setting would be informative (Enquist and Leib, 2016). The natural route of HSV-1 infection occurs through retrograde transport from the eye to the CNS, where the cGAS-STING pathway is activated following phagocytosis of infected neurons by microglia to control neuroinvasive strains of HSV-1 (Reinert et al., 2016). cGAS and STING are indispensable in controlling the virus in this setting. Thus, it will be of interest to determine how hnRNPA2B1 functions in more physiologically relevant routes of infection. Comparisons of hnRNPA2B1 to cGAS are also instantly drawn by the ability of both proteins to recognize not only pathogen-derived but also host DNA. Failure to control DNA accumulation drives a range of debilitating human diseases,
Molecular Cell
Spotlight
Figure 1. hnRNPA2B1 Amplifies the cGAS-STING Pathway during DNA Virus Infection (A) Demethylation, dimerization, and translocation of hnRNPA2B1 following detection of viral DNA. (B) Association and activation of TBK1 by hnRNPA2B1. (C) hnRNPA2B1 mediated m6A of cGAS, STING, and IFI16 mRNA.
such as Aicardi-Goutie`res syndrome (AGS), resulting from inactivating mutations in nucleases such as TREX1. Indeed, cGAS plays a critical role in recognizing the accumulated DNA, driving the pathophysiology of this condition (Gao et al., 2015; Mackenzie et al., 2017). Thus, it will be interesting to determine if hnRNPA2B1 sensing of host DNA also contributes to the pathophysiology of AGS and related conditions. In support of this, it is intriguing that autoantibodies to hnRNPA2B1 have been detected in patients with lupus (Dumortier et al., 2000) This study also expands on the importance of PTMs in the positive and negative regulation of DNA sensors. Methylation sequesters monomeric hnRNPA2B1 in the nucleus, whereas acetylation can control activity and cellular localization of cGAS and IFI16. Indeed, nuclear localization, cytoplasmic translocation, and epigenetic modifications are characteristics encompassing multiple DNA sensors including cGAS, IFI16/p204, and now hnRNPA2B1 (Dai et al., 2019; Li et al., 2012).
These findings expand the spatial regulation of DNA sensors to include sensors in the endosome (TLR9), cytosol (cGAS/ AIM2), and now the nucleus (hnRNPA2B1 and cGAS). The work also highlights the temporal regulation of antiviral pathways with distinct sensors affecting the priming and amplification of these pathways. cGAS, IFI16, and now hnRNPA2B1 form a triad of nuclear DNA sensors that collectively synergize to amplify STING activation and restrict HSV-1 replication. Future work investigating cross-talk between these receptors and their role in different cell types may uncover further layers of regulation of antiviral immunity. Lastly, this study also raises questions regarding the juxtaposition of nuclear and cytoplasmic DNA sensing and highlights how transfer of secondary signals across the nuclear membrane is an essential component of DNA sensing. Indeed, if cGAS is truly a nuclear protein, demonstrating nucleocytoplasmic cGAMP trafficking will be important. The study from Cao and colleagues illuminates new complexity to
DNA sensing and sheds new light on the communication between nuclear sensors and cytoplasmic effectors. ACKNOWLEDGMENTS This work is supported by grants from the NIH (AI128358 and AI067497).
REFERENCES Carpenter, S., Aiello, D., Atianand, M.K., Ricci, E.P., Gandhi, P., Hall, L.L., Byron, M., Monks, B., Henry-Bezy, M., Lawrence, J.B., et al. (2013). A long noncoding RNA mediates both activation and repression of immune response genes. Science 341, 789–792. Dai, J., Huang, Y.-J., He, X., Zhao, M., Wang, X., Liu, Z.-S., Xue, W., Cai, H., Zhan, X.-Y., Huang, S.-Y., et al. (2019). Acetylation Blocks cGAS Activity and Inhibits Self-DNA-Induced Autoimmunity. Cell 176, 1447–1460.e14. Dumortier, H., Monneaux, F., Jahn-Schmid, B., Briand, J.-P., Skriner, K., Cohen, P.L., Smolen, J.S., Steiner, G., and Muller, S. (2000). B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice. J. Immunol. 165, 2297–2305.
Molecular Cell 76, October 3, 2019 9
Molecular Cell
Spotlight Enquist, L.W., and Leib, D.A. (2016). Intrinsic and Innate Defenses of Neurons: De´tente with the Herpesviruses. J. Virol. 91, e01200–e01216. Gao, D., Li, T., Li, X.-D., Chen, X., Li, Q.-Z., WightCarter, M., and Chen, Z.J. (2015). Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. USA 112, E5699–E5705. Li, T., Diner, B.A., Chen, J., and Cristea, I.M. (2012). Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc. Natl. Acad. Sci. USA 109, 10558–10563.
10 Molecular Cell 76, October 3, 2019
Liu, D., Wu, H., Wang, C., Li, Y., Tian, H., Siraj, S., Sehgal, S.A., Wang, X., Wang, J., Shang, Y., et al. (2019). STING directly activates autophagy to tune the innate immune response. Cell Death Differ. 26, 1735–1749. Mackenzie, K.J., Carroll, P., Martin, C.-A., Murina, O., Fluteau, A., Simpson, D.J., Olova, N., Sutcliffe, H., Rainger, J.K., Leitch, A., et al. (2017). cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465. Reinert, L.S., Lopusna´, K., Winther, H., Sun, C., Thomsen, M.K., Nandakumar, R., Mogensen, T.H., Meyer, M., Vægter, C., Nyengaard, J.R.,
et al. (2016). Sensing of HSV-1 by the cGASSTING pathway in microglia orchestrates antiviral defence in the CNS. Nat. Commun. 7, 13348. Wang, L., Wen, M., and Cao, X. (2019). Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science 365, eaav0758. Zierhut, C., Yamaguchi, N., Paredes, M., Luo, J.D., Carroll, T., and Funabiki, H. (2019). The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death. Cell 178, 302–315.e23.