Robert Silverman, Ph.D.
The Mal and Lea Bank Chair
Lerner Research Institute,
9500 Euclid Avenue, Cleveland, Ohio 44195
Our laboratory probes fundamental innate immune responses that profoundly impact viral infections and cancer. Interferons (IFNs) are critically important cytokines that control innate immunity against viruses and cancer. IFNs exert their antiviral effects in part through 2’,5’-oligoadenylate (2-5A) synthetase (OAS)-RNase L, the focus of my research for the past 40 years. OAS-RNase L is now generally recognized as one of the principal intracellular mechanisms of antiviral innate immunity. Viruses produce double-stranded (ds) RNAs that activate IFN-inducible OAS enzymes to produce 2-5A from ATP. 2-5A binds to the ubiquitous, but latent, RNase L, causing its dimerization and activation. Once activated, RNase L cleaves viral and cellular RNA, virus replication is inhibited and cell death occurs by apoptosis. We were the first to purify and clone RNase L and to establish the antiviral activity of RNase L in vivo using genetically deficient mice. We later showed that RNase L contributes to viral-mediated inflammation through the NLPR3 inflammasome. Our lab was also the first to show that self-RNA, cleaved by RNase L, could trigger a type I IFN response. In collaboration with Frank Sicheri (Toronto), a detailed and intricate structure of dimeric RNase L in a complex with 2-5A and ATP was revealed. With Susan R. Weiss (Philadelphia), we uncovered how certain coronaviruses (MHV and MERS-CoV) and rotaviruses evade the IFN system by encoding phosphodiesterases (PDEs) that degrade 2-5A thus preventing RNase L activation. In one study, mutation of the MHV PDE (ns2) impaired virus replication in wild type mice, but the mutant virus infected normally in mice lacking RNase L. Those studies demonstrated the ability of OAS-RNase L to control a coronavirus infection in vivo.
Recently we showed that uninfected cells sometimes produce dsRNA that triggers the OAS-RNase L pathway. We found that dsRNA transcribed from repetitive regions of the genome can activate OAS enzymes. Drugs that cause epigenetic modification of DNA, such as 5-azacytidine (AZA), are used clinically to treat myelodysplastic syndromes and acute myeloid leukemia. In addition, AZA is being investigated for use against a range of different types of solid tumors, including lung cancer. Treatment with AZA causes demethylation of DNA, thus increasing RNA synthesis, including the synthesis of dsRNA, which is otherwise produced in virus-infected cells. We determined that cell death in response to AZA requires the OAS-RNase L pathway, with implications for the control of certain types of cancer.
These investigations have implications for virus-mediated pathology and human survival from viral pathogens and cancer. Dr. Silverman has investigated the role interferons (IFNs) play in protecting the body from viruses and cancer for the past 40 years. IFNs are proteins made by and secreted from host cells in response to the presence of certain pathogens. Dr. Silverman is particularly interested in how the enzyme system OAS-RNase L, which protects higher vertebrates from both RNA and DNA viruses, contributes to this process. As often happens, however, viruses have adapted and evolved to inhibit OAS-RNase L activity. He continues on in his decades-long quest to better understand how exactly RNase L works on a cellular and molecular level. This knowledge may help researchers prevent harmful viruses from evading the protective effects of RNase L, and thereby mitigate viral infections and their serious effects. Recent studies implicate the OAS-RNase L pathway in the anti-tumor cell effects of the DNA demethylating drug, 5-azacytidine (AZA), providing a more comprehensive understanding about how AZA and dsRNA prevent the proliferation of cancer cells. This research offers important insights about how a host antiviral protein can, under some conditions, function against cancer cells.
The intersection between innate immunity and tumor suppression is the focus of the Silverman Lab. Our studies probe fundamental molecular and cellular processes that impact viral infections and cancer. We seek a better understanding of how the mammalian cell resists viral infections and how the virus antagonizes the host response to infection.
The interferon (IFN) system is a frontline host defense against invading viral pathogens. The 2',5'-oligoadenylate (2-5A)synthetase (OAS)-RNase L pathway is among the most potent IFN-induced antiviral effectors, blocking viral infections by several mechanisms, including directly cleaving viral single-stranded RNA genomes, depleting viral and host mRNA available for translation, and enhancing type I IFN induction. Type I IFNs bind to the cell surface receptor IFNAR initiating JAK-STAT signaling to the OAS genes, which results in elevated levels of OAS proteins. When activated by viral double-stranded (ds)RNA, certain OAS isoforms use ATP to synthesize 5'-triphosphorylated 2-5A. Trimer and longer species of 2-5A bind with high specificity and affinity to the inactive monomeric RNase L, causing it to dimerize and become active.
Antiviral Mechanisms of 2-5A Dependent RNase L:
RNase L is a principal mediator of the innate antiviral response and is thus critically important for human health. Previously, we purified RNase L(JBC, 1988, PMID: 3366783), cloned RNase L (Cell, 1993, PMID: 7680958), and knocked out the RNase L gene in mice (EMBO J, 1997, PMID: 9351818). We showed that RNA cleavage products of self-RNA produced by RNase L function as PAMPs in amplifying innate immune signaling (Nature, 2007, PMID: 17653195) and inflammation (Cell, Host & Microbe, 2015, PMID: 25816776). Our long-term objectives are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles, spread and pathogenesis. The knowledge to be gained from these studies may contribute to improved treatments for viral infections and cancer.
Control of Viral Pathogenesis by Regulation of 2-5A Levels (a collaborative project with Susan Weiss, University of Pennsylvania):
The ability of viruses to evade the IFN antiviral response plays an important role in viral tropism and disease pathogenesis. Relevant to this project, the murine coronavirus ns2 protein has 2’,5’-phosphodiesterase (PDE) activity that antagonizes the IFN regulated OAS-RNase L antiviral pathway by eliminating 2-5A thereby promoting hepatitis; several other viruses and host cells have proteins with similar activities (Cell Host & Microbe, 2012, PMID: 22704621). With the teams of Susan Weiss and John Patton (NIAID, NIH), we reported group A rotavirus, an important cause of acute gastroenteritis in children worldwide, encodes a similar 2’,5’-PDE (PNAS, 2013, PMID: 23878220). We showed the NS4b protein of MERS coronavirus has a similar 2-5A degrading activity (mBio, 2016, PMID: 27025250). We also showed that the mammalian AKAP7 protein is a related PDE for 2-5A (mBio, 2014, PMID: 24987090). The discovery and use of inhibitors of viral and cellular PDEs to enhance RNase L activity selectively in virus-infected cells potentially provides a novel mode of regulation of the antiviral response, with broad implication for the control of viral infections.
OAS-RNase L Innate Immune Pathway Mediates the Cytotoxicity of a DNA-demethylating Drug:
Drugs that cause epigenetic modification of DNA, such as 5-azacytidine (AZA), are used clinically to treat myelodysplastic syndromes and acute myeloid leukemia. In addition, AZA is being investigated for use against a range of different types of solid tumors, including lung and colorectal cancers. Treatment with AZA causes demethylation of DNA, thus increasing RNA synthesis, including the synthesis of double-stranded RNA, which is otherwise produced in virus-infected cells. We recently determined that cell death in response to AZA requires the antiviral enzyme RNase L (PNAS, 2019, PMID: 30814222). The results identify a drug target for enhancing the anticancer activity and reducing the toxicity of AZA and related drugs.
Selected from 249 publications:
Silverman, R.H. and Atherly, A.G. The search for ppGpp and other unusual highly phosphorylated nucleotides in eukaryotes. Microbiological Reviews 43, 27-41, 1979.
Silverman, R.H., Cayley, P.J., Knight, M., Gilbert, C.S., and Kerr, I.M. Control of the ppp(A2'p)nA system in HeLa cells: effects of interferon and virus infection. Eur. J. Biochem. 124, 131-138, 1982.
Silverman, R.H., Watling, D., Balkwill, F.R., Trowsdale, J., and Kerr, I.M. The ppp(A2'p)nA and protein kinase systems in wild type and interferon-resistant Daudi cells. Eur. J. Biochem. 126, 333-341, 1982.
Silverman, R.H., Skehel, J.J., James, T.C., Wreschner, D.H., and Kerr, I.M. Ribosomal RNA cleavage as an index of ppp(A2'p)nA activity in interferon-treated encephalomyocarditis virus-infected cells. J. Virol. 46, 1051-1055, 1983.
Silverman, R.H., Jung, D.D., Nolan-Sorden, N.L., Dieffenbach, C.W., Kedar, V.P., and SenGupta, D. Purification and analysis of murine 2-5A-dependent RNase. J. Biol. Chem. 263, 7336-7341, 1988.
Zhou, A., Hassel, B.A., and Silverman, R.H. Expression cloning of 2-5A-dependent RNase-a uniquely regulated mediator of interferon action. Cell, 72, 753-765, 1993.
Hassel, B.A., Zhou, A., Sotomayor, C., Maran, A., and Silverman, R.H. A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J., 12, 3297-3304, 1993.
Zhou, A., Paranjape, J., Brown, T.L., Nie, H., Naik, S., Dong, B., Chang, A., Trapp, B. Fairchild, R., Colmenares, C., and Silverman, R.H. Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate dependent RNase L. EMBO J. 16, 6355-6363,1997.
Der, S., Zhou, A., Williams, B.R.G., and Silverman, R.H.Identification of Genes Differentially Regulated by IFN-α β or γ or Using Oligonucleotide Arrays.Proc. Natl. Acad. Sci. U.S.A 95,15623-15628, 1998.
Zhou, A., Paranjape, J.M., Der, S.D., and Williams, B.R.G. and Silverman, R.H. Novel Innate Mechanisms of Interferon Action are Revealed in Triply Deficient Mice. Virology, 258, 435-440, 1999.
Carpten, J., N., et al. Germline Mutations in the Ribonuclease L (RNase L) Gene in Hereditary Prostate Cancer 1 (HPC1) -Linked Families. Nature Genetics, 30, 181-184, 2002.
Casey, G., et al.RNASEL R462Q variant is implicated in 13% of prostate cancer cases. Nature Genetics, 32, 581-583, 2002. Silverman, R.H. Implications for RNase L in Prostate Cancer Biology.Biochemistry, 42, 1805-1812, 2003.
Xiang, Y., Wang,Z., Murakami, J., Plummer, S., Klein, E.A., Carpten, J., Trent, J., Isaacs W., Casey, G., and Silverman, R. H. Effects of RNase L mutations associated with prostate cancer on apoptosis induced by 2',5'-oligoadenylates. Cancer Res. 63: 6795-6801, 2003.
Malathi, K., Paranjape, J.M., Bulanova, E., Shim, M., Guenther-Johnson, J.M., Faber, P.W., Eling, T.E., Williams, B.R.G., and Silverman, R.H. A novel transcriptional signaling pathway in the interferon system mediated by 2'-5'-oligoadenylate activation of RNase L. Proc. Natl. Acad. Sci. U.S.A., 102, 14533-14538, 2005.
Malathi, K., Dong, B., Gale, M., and Silverman, R.H. Small Self RNA Generated by RNase L Amplifies Antiviral Innate Immunity. Nature, 448: 816-819, 2007.
Malathi, K., Saito, T., Crochet, N., Barton, D.J.,Gale, M.and Silverman, R.H. RNase L Releases a Small RNA from HCV RNA that Refolds into a Potent PAMP. RNA, 16: 2108-2119, 2010.
Jha, B.K., Polyakova, I., Kessler, P., Dong, B., Dickerman, B., Sen, G.C., and Silverman, R.H. Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity. J. Biol. Chem.,286: 26319-26326. 2011.
Zhao, L., Jha, B., Wu, A., Elliott, R., Ziebuhr, J., Gorbalenya, A.E., Silverman, R.H., Weiss, S.R. 2012. Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology. Cell Host and Microbe, 11:607-616, 2012.
Chakrabarti, A., Ghosh, P.K., Banerjee, S., Gaughan, C., and Silverman, R.H. RNase L triggers autophagy in response to viral infections. J. Virol. 86: 11311-21, 2012.
Jha, B.K., Dong, B., Nguyen, C.T., Polyakova, I. and Silverman, R.H. Suppression of antiviral innate immunity by sunitinib enhances oncolytic virotherapy. Molecular Therapy, 21:1749-57, 2013.
Zhao, L., Birdwell, D. Wu, A., Elliott, R., Rose, K., Phillips, J., Li, Y., Grinspan, J., Silverman, R., and Weiss, S. Cell type specific activation of the OAS-RNase L pathway by a murine coronavirus. J. Virol., 87:8408-18, 2013. [Featured article in "JVI Spotlight"]
Sorgeloos, F., Jha, B.K., Silverman, R.H., and Michiels, T.Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L. PLoS Pathogens, 9(6):e1003474, 2013.
Zhang, R., Jha, B.K., Ogden, K., Dong, B., Zhao, L., Elliot, R., Patton, J.T., Silverman, R.H.* and Weiss, S.R.* Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc. Natl. Acad. Sci., 110:13114-9, 2013. [*Co-corresponding authors].
Zhou, Y., Kang, M.J., Silverman, R.H., Lee, C.G., and Elias, J.A. Role of RNase L in viral pathogen-associated molecular pattern/influenza virus and cigarette smoke-induced remodeling. J. Immunol., 191:2637-46, 2013.
Zhang, A., Dong, B., Doucet, A.J., Moldovan, J.B., Moran, J.V., and Silverman, R.H. RNase L restricts the mobility of engineered retrotransposons in cultured human cells. Nucleic Acids Research, 42: 3803-20, 2013.
Huang, H., Zeqiraj, E., Dong, B., Jha, B.K., Duffy, N., Orlicky, S., Thevakumaran, M., Pillon, M.C., Ceccarelli, D.F., Wan, L., Juang, Y.C., Mao, D.Y.L., Gaughan, C., Brinton, M.A., Perelygin, A.A., Kourinov, I., Guarne, A., Silverman, R.H.*, Sicheri,F.* Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity. Molecular Cell 53(2):221-34, 2014. [*Co-corresponding authors] [highlighted as an Editor’s Select commentary in Cell, 2014, “The Fantastic RNase L”]
Silverman, R.H. and Weiss, S.R. Viral phosphodiesterases that antagonize double strand RNA signaling to RNase L by degrading 2-5A. J. Interferon & Cytokine Res., 34(6): 455-463, 2014., 2014.
Cooper, D.A., Jha, B.K., Silverman, R.H., Hesselberth, J.R., and Barton, D.J. Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. Nucleic Acids Res., 42:5202-16, 2014.
Banerjee, S., Chakrabarti, A., Jha, B.K., Weiss, S.R., and Silverman, R.H. Cell type-specific effects of RNase L on viral induction of IFN-. mBio, 5(2): e00856-14, 2014.
Gusho, E., Zhang, R., Jha, B.K., Thornbrough, J.M., Dong, B., Gaughan, C., Elliott, R., Weiss, S.R. and Silverman, R.H. Murine AKAP7 has a 2’,5’-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene. mBio,34:455-63, 2014.
Franchi, L., Eigenbrod, T., Munoz-Planillo, Ozkurede, U., Kim, Y.G., Chakarbarti, A., Gale, M., Silverman, R.H., Colonna, M., Akira, S., Nunez, G. Cytosolic double-stranded RNA activates the NLRP3 inflammasome via MAVS-induced membrane permeabilization and K+ efflux. J. Immunol. 193, 4214-4222. 2014.
Cooper, D., Banerjee, S., Chakrabarti, A., Garcia-Sastre, A., Hesselberth, J., Silverman, R.H., Barton, D.J. Ribonuclease L targets distinct sites in influenza A virus RNAs. J. Virol. 89, 2764-2776, 2015.
Chakrabarti, A., Banerjee, S., Franchi, L., Loo, Y.M., Gale, M., Nunez, G., Silverman, R.H. RNase L activates the NLRP3 inflammasome during viral infections. Cell Host & Microbe, 17: 466-477, 2015 [On the Cover].
Silverman, R.H. Caps off to poxviruses. Cell Host & Microbe, 17: 2878-2879, 2015.
Ogden, K.M., Hu, L., Jha, B.K., Sankaran, B., Weiss, S.R., Silverman, R.H., Patton, J.T., and Prasad, B.V.V. Structural basis for 2¢-5¢-oligoadenylate binding and enzyme activity of a viral RNase L antagonist. J. Virol., 89: 6633-6645, 2015.
Banerjee, S., Li, G., Li, Y., Gaughan, C., Baskar, D., Parker, Y., Lindner, D.J., Weiss, S.R., and Silverman, R.H. RNase L is a negative regulator of cell migration. Oncotarget, 6: 44360-44372, 2015.
Birdwell, L.D., Zalinger, Z.B., Li, Y., Wright, P.W., Elliott, R., Rose, K.M., Silverman, R.H., Weiss,. S.R. Activation of RNase L by murine coronavirus in myeloid cells is dependent on basal Oas gene expression and independent of virus-induced interferon. J. Virology, 90:3160-72, 2016.
Sui, B., Huang, J., Jha, B.K., Yin, P., Zhou, M., Fu, Z.F., Silverman, R.H., Weiss, S.R., Peng, G., and Zhao, L. Crystal structure of the mouse hepatitis virus ns2 phosphodiesterase domain that antagonizes RNase L activation. J. gen. Virol., 97:880-886, 2016.
Li, Y., Banerjee, S., Wang, Y., Goldstein, S.A., Dong, B., Gaughan, C. *Silverman, R.H., and *Weiss, S.R. Activation of RNase L is dependent on OAS3 expression during infection with diverse viruses. Proc. Natl. Acad. Sci., 113: 2241-6, 2016. [*Co-corresponding authors]
Thornbrough, J.M., Jha, B.K., Yount, B., Goldstein, S.A., Li, Y., Elliott, R., Sims, A.C., Baric, R.S., Silverman, R.H., and Weiss, S.R. Middle east respiratory syndrome coronavirus NS4b protein inhibits host ribonuclease L activation. mBio, 7(2). pii: e00258-16, 2016.
Goldstein, S.A., Thornbrough, J.M., Zhang, R., Jha, B.K., Li, Y., Elliott, R., Quiroz-Figueroa, Chen, A.I., Silverman, R.H., and Weiss, S.R. Lineage A Betacoronavirus NS2 proteins and homologous Torovirus Berne pp1a- carboxyterminal domain are phosphodiesterases that antagonize activation of RNase L. J. Virol., Feb 14;91(5). pii: e02201-16. doi: 10.1128/JVI.02201-16, 2017.
Kindler, E., Gil-Cruz, C., Spanier, J., Li, Y., Wilhelm, J., Rabouw, H., Zust, R., Hwang, M. V’kovski, P., Stalder, H. Marti, S., Habjan, M., Cervantes-Barragan, L., Elliot, R., Karl, M., Gaughan, C., van Kuppeveld, F., Silverman, R.H., Keller, M., Ludewig, B., Bergmann, C., Ziebuhr, Weiss, S.R., Kalinke, U., Thiel, V. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathogens Feb 3;13(2):e1006195. doi: 10.1371/journal.ppat.1006195, 2017.
Li, Y., Banerjee, S., Goldstein, S., Dong, B., Gaughan, C., Rath, S., Donovan, J., Korennykh, A., Silverman, R.H.*, Weiss, S.R.* Ribonuclease L mediates the cell-lethal phenotype of the double-stranded RNA editing enzyme ADAR1 in a human cell line. eLife, Elife. Mar 31;6. pii: e25687. doi: 10.7554/eLife.25687, 2017. [*Co-corresponding authors].
Drappier, M., Jha, B.K., Stone, S., Elliott, R., Zhang, R., Vertommen, D., Weiss, S.R., Silverman, R.H., Michiels, T. A novel mechanism of RNase L inhibition: Theiler’s virus L* protein prevents 2-5A from binding to RNase L. PLoS Pathogens, 14(4):e1006989, 2018.
Tang, W., Wallace T.A., Yi, M., Magi-Galluzzi, C., Dorsey, T.H., Onabajo, O.O., Obajemu, A., Jordan, S.V., Loffredo, C.A., Stephens, R.M., Silverman, R.H., Stark, G.R., Klein, E.A., Prokunina-Olsson, L., Ambs, S. IFNL4-ΔG allele is associated with an interferon signature in tumors and survival of African-American men with prostate cancer. Clin Cancer Res. 24: 5471-5481, 2018.
Banerjee, S., Gusho, E., Gaughan, Dong, B., Gub, X., Holvey-Bates, E., Talukdar, M., Li, Y., Weiss, S.R., Sicheri, F., Saunthararajah, Y., Stark, G.R., Silverman, R.H. The OAS-RNase L innate immune pathway mediates the cytotoxicity of a DNA demethylating drug. Proc. Natl. Acad. Sci., 116:5071-5076, 2019.
Whelan, J.N., Li, W., Silverman, R.H., and Weiss, S.R. Zika Virus Production Is Resistant to RNase L Antiviral Activity. J. Virol. Jul 30;93(16):e00313-19. doi: 10.1128/JVI.00313-19. 2019.
Li, Y., Dong, B., Wei, Z., Silverman, R.H., Weiss, S.R. Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling. mBio Nov 12;10(6):e02414-19. doi: 10.1128/mBio.02414-19. 2019.
Song, Y., Feng , N., Sanchez-Tacuba, L., Yasukawa, L.L., Ren, L., Silverman, R.H., Ding, S., Greenberg, H.B. Reverse Genetics Reveals a Role of Rotavirus VP3 Phosphodiesterase Activity in Inhibiting RNase L Signaling and Contributing to Intestinal Viral Replication In Vivo. J. Virol. Apr 16;94(9):e01952-19. doi: 10.1128/JVI.01952-19. 2020.
Kondratova, A.A., Cheon, H., Dong, B., Holvey-Bates, E.G., Hasipek, M., Taran, I., Gaughan, C., Jha, B.K., Silverman, R.H., and Stark, G.R. Suppressing PARylation by 2',5'-oligoadenylate Synthetase 1 Inhibits DNA Damage-Induced Cell Death. EMBO J. Jun 2;39(11):e101573. doi: 10.15252/embj.2019101573. 2020.
Bergmann, C.C., and Silverman, R.H. COVID-19: Coronavirus Replication, Pathogenesis, and Therapeutic Strategies. Cleve. Clin. J. Med. Jun;87(6):321-327. doi: 10.3949/ccjm.87a.20047. 2020.