Interferons (IFNs) are a group of cytokines that play essential roles in mediating innate immunity to a wide range of different types of viruses. The anti-viral activities of IFNs are due, in part, to RNA degradation by RNase L, a remarkable enzyme that is activated by 2',5' -oligoadenylates (2-5A). 2-5A is produced by a family of IFN-inducible synthetases that require double-stranded RNA (dsRNA), often of viral origin, for their activity. Interestingly, RNase L deficient mice, generated during this project, have enhanced susceptibility to lethal viral infections and are deficient in spontaneous and induced apoptosis. Our preliminary data further implicate 2-5A and RNase L as the mediators of a specific ribotoxic stress response that could result in the elimination of virus-infected cells. In this proposal, we aim to elucidate the specific role of the 2-5A/RNase L system in the overall biologic response to IFN.
Our specific aims i nclude to: (1) probe the function and regulation of RNase L by constructing chimeras between RNase L and the related nuclease Ire1; use two-hybrid systems and mass spectrometry to clone and identify host and vaccinia virus proteins that interact with and regulate RNase L; and perform structural studies on RNase L by NMR and crystallography; (2) determine how RNase L causes its anti-viral and pro-apoptotic activities by identifying the RNA substrate preferences of RNase L with DNA microarrays; investigate how RNase L contributes to dsRNA-activation of stress activated protein kinase JNK, and probe the apoptotic pathway initiated by 2-5A activation of RNase L; and (3) investigate the biologic actions of RNase L by studying involvement of RNase L in cellular immunity; determine the role of RNase L in resistance to HSV-1 infections, and dissect RNase L mediated responses to picomavirus infections in cultured primary cardiac myocytes and in organs of infected mice. RNase L is an important mediator of the anti-viral and apoptotic activities of IFNs. Activation of RNase L by a small and unique molecule, 2-5A, provides an opportunity to control viral infections at the level of RNA turnover. Therefore, continuation of this PROJECT is expected to lead to the eventual development of novel strategies for treatment of viral infections and cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA044059-17
Application #
6430602
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1986-04-01
Project End
2007-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
17
Fiscal Year
2002
Total Cost
$352,165
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Li, Yize; Banerjee, Shuvojit; Goldstein, Stephen A et al. (2017) Ribonuclease L mediates the cell-lethal phenotype of double-stranded RNA editing enzyme ADAR1 deficiency in a human cell line. Elife 6:
Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia et al. (2017) Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathog 13:e1006195
Birdwell, L Dillon; Zalinger, Zachary B; Li, Yize et al. (2016) Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on Basal Oas Gene Expression and Independent of Virus-Induced Interferon. J Virol 90:3160-72
Li, Yize; Banerjee, Shuvojit; Wang, Yuyan et al. (2016) Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses. Proc Natl Acad Sci U S A 113:2241-6
Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam et al. (2015) RNase L targets distinct sites in influenza A virus RNAs. J Virol 89:2764-76
Banerjee, Shuvojit; Li, Geqiang; Li, Yize et al. (2015) RNase L is a negative regulator of cell migration. Oncotarget 6:44360-72
Chakrabarti, Arindam; Banerjee, Shuvojit; Franchi, Luigi et al. (2015) RNase L activates the NLRP3 inflammasome during viral infections. Cell Host Microbe 17:466-77
Huang, Hao; Zeqiraj, Elton; Dong, Beihua et al. (2014) Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity. Mol Cell 53:221-34
Silverman, Robert H; Weiss, Susan R (2014) Viral phosphodiesterases that antagonize double-stranded RNA signaling to RNase L by degrading 2-5A. J Interferon Cytokine Res 34:455-63
Banerjee, Shuvojit; Chakrabarti, Arindam; Jha, Babal Kant et al. (2014) Cell-type-specific effects of RNase L on viral induction of beta interferon. MBio 5:e00856-14

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