RNase L is a principle mediator of the innate antiviral response and is thus critically important for human health. Virus replication in higher vertebrates is restrained by interferons (IFN) that cause cells to transcribe antiviral genes, including genes for 2',5'-oligoadenylate synthetases (OAS), a group of pathogen recognition receptors (PRR). The viral pathogen associated molecular pattern (PAMP), double-stranded RNA, activates OAS to produce 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) whose function is to stimulate RNase L. The OAS-RNase L system is a classical innate immune pathway that both responds to and generates RNA PAMPs to produce a broadly active antiviral response. In preliminary studies we show that activation of RNase L during viral infections initiates RIG-I like receptor (RLR) signaling, inflammasome activation, and autophagy. Some of these events are triggered by small, highly structured RNAs (called suppressor of virus RNA or svRNA) produced when viral and cellular single stranded RNAs are cleaved by RNase L. Our long-term objectives are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles, spread and pathogenesis. Our hypothesis is that RNase L blocks virus replication by directly cleaving viral and cellular ssRNA, and indirectly by triggering RLR signaling, inflammasome activation, and autophagy. In the proposed project, we seek to raise our knowledge about a key antiviral pathway to the next level.
Our Specific Aims are: (1) To investigate how RNase L counteracts viral infections through RLR signaling, we will use deep sequencing to identify RIG-I bound small RNAs produced by RNase L in a cell-free system and in intact virus-infected cells, synthesize and characterize svRNAs for the ability to activate RIG-I and induce IFN-?, and determine the sequence and structural characteristics of svRNAs that result in RIG-I activation. (2) To investigate how RNase L activates inflammasomes during viral infections, we will identify the PRRs that interact with svRNAs by immunoblotting and mass spectrometry, and we will use gene knockout cells to determine how inflammasome signaling is regulated by RNase L. (3) To investigate the role of RNase L in autophagy during viral infections, we will determine the impact of rRNA cleavage by RNase L in ribosomes on autophagy by controlling access of the ribosome to RNase L, study how RNase L induction of autophagy impacts RLR- and inflammasome-signaling in gene knockout cells, and determine how regulation of autophagy by RNase L affects virus replication and innate immune signaling in wild type and mutant mice. The proposed research on RNase L could eventually provide the basis for developing a broad-spectrum antiviral drug. In addition, these studies could lead to strategies for the treatment or prevention of cancers caused by oncogenic viruses. Therefore, there are cogent and health-related justifications for these studies.
Antiviral innate immunity is mediated, in part, by the host protein RNase L which cleaves both viral and cellular RNA. In these studies, we will investigate how RNase L amplifies production of antiviral cytokines and how it eliminates virus particles through a process known as autophagy. The knowledge to be gained from the proposed studies could contribute to improved treatments for viral infections.
|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|
|Cooper, Daphne A; Jha, Babal K; Silverman, Robert H et al. (2014) Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. Nucleic Acids Res 42:5202-16|
|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|
|Franchi, Luigi; Eigenbrod, Tatjana; Muñoz-Planillo, Raúl et al. (2014) Cytosolic double-stranded RNA activates the NLRP3 inflammasome via MAVS-induced membrane permeabilization and K+ efflux. J Immunol 193:4214-22|
|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|
|Zhang, Ao; Dong, Beihua; Doucet, Aurelien J et al. (2014) RNase L restricts the mobility of engineered retrotransposons in cultured human cells. Nucleic Acids Res 42:3803-20|
|Sorgeloos, Frederic; Jha, Babal Kant; Silverman, Robert H et al. (2013) Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L. PLoS Pathog 9:e1003474|
|Zhou, Yang; Kang, Min-Jong; Jha, Babal Kant et al. (2013) Role of ribonuclease L in viral pathogen-associated molecular pattern/influenza virus and cigarette smoke-induced inflammation and remodeling. J Immunol 191:2637-46|
|Zhao, Ling; Birdwell, L Dillon; Wu, Ashley et al. (2013) Cell-type-specific activation of the oligoadenylate synthetase-RNase L pathway by a murine coronavirus. J Virol 87:8408-18|
|Li, Sam X; Barrett, Bradley S; Harper, Michael S et al. (2013) Ribonuclease L is not critical for innate restriction and adaptive immunity against Friend retrovirus infection. Virology 443:134-42|
Showing the most recent 10 out of 92 publications