Research in the Krishnamurthy lab focuses on host response during viral infections. Our studies address the fundamental mechanisms by which cells resist infection and how cells know they are infected by a virus. The innate immune pathway is the first line of defense against viruses and functions to limit viral replication and spread. Pattern-Recognition Receptors including Rig-I-like helicases (Rig-I and MDA5) and Toll-like receptors (TLRs) recognize conserved microbial features (Pathogen associated Molecular Pattern, PAMP) and provide signals to initiate immune response by producing type I IFN and cytokines. Double stranded RNA produced during viral infections serves as PAMP and activates the IFN-inducible 2’,5’ - oligoadenylate synthetase (OAS) which converts cellular ATP to unique 2’,5’ - linked oligoadenylates, 2-5A, which binds and activates a ubiquitous and latent endoribonuclease, RNase L. Activated RNase L cleaves single stranded viral and host RNAs to produce small RNAs with duplex  structures which can signal through Rig-I and MDA5 to amplify the production of IFNb. Studies in the lab are aimed at investigating the signaling pathway initiated by small RNA cleavage products of RNase L and the expanding roles of RNase L in innate immunity. Areas of research in our group include:

A. Innate immune strategies during viral infections using human and murine models

RNase L-induced antiviral Stress Granules (SG) during viral infection: Virus infection leads to activation of the interferon-induced endoribonuclease, RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRR) like Retinoic acid-inducible I (Rig-I) and or melanoma differentiation-associated protein 5 (MDA5) to further amplify interferon (IFN) production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how the cells coordinate RNA sensing to signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce formation of antiviral stress granule (avSG) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), and recruit antiviral proteins Rig-I, PKR, OAS and RNase L to avSG.

Regulation of autophagy and cell death pathways: We have shown recently that activation of RNase L induces autophagy involving the activities of dsRNA-dependent protein kinase R (PKR) and c-jun N-terminal kinase (JNK). ). The regulation of autophagy and innate immunity to viral infections appears to involve overlapping signaling pathways and autophagy-related proteins can have both proviral and antiviral effects. It is not clear how these two pathways interact and how the activation of one might complicate the outcome of the other, i.e., could the induction of autophagy modulate innate immune responses to viral infection?  Ongoing studies are determining the role of RNA signaling pathways in the crosstalk between autophagy and apoptosis during viral infections.

Non-enzymatic antiviral role of RNase L: Our recent studies identified a novel interaction between the antiviral endoribonuclease RNase L and the actin binding protein Filamin A that enhances host defense by preventing viral entry into naive cells. This role for RNase-L is independent of its enzymatic function. Virus infection alters actin dynamics, disrupts the RNase L-Filamin A complex and releases RNase L to mediate antiviral signaling and effector functions via its established nucleolytic activitiesCurrent studies are aimed at determining the impact of altered actin dynamics and cytoskeletal proteins in regulating virus trafficking in polarized and non-polarized models of infection.

Role of small cellular RNAs generated by RNaseL in innate immunity: Previous studies showed that activity of RNase L on cellular RNA generates small self-RNAs that activate Rig-I and MDA5, producing IFN-b through activation of the IRF3 transcription factor. Cloning and characterization of various host small RNAs will allow us to establish the features of RNAs that are needed to activate Rig-I or MDA5 and the basis of discrimination of “self” and “nonself”. The identification of small RNAs with ability to induce IFN-b and or modulate inflammatory responses would be a critical early step in the development of antiviral and antitumor therapies with improved outcomes.

RNase L-Filamin A interaction in prostate cancer: We have identified a novel interaction of RNase L with actin-binding protein, Filamin A. Interaction of Filamin A with AR inhibits transcriptional activity of AR. Previous studies have shown that RNase L interacts with AR, and the interaction is stimulated by androgens. Also, mutations in RNASEL correlate with incidence of hereditary prostate cancer. In light of these correlations, we are investigating the role of RNase L in prostate cancer.

 B. Innate immune strategies during viral infections using fish models

Immune evasion in Aquatic Rhabdoviral Pathogens: Another area of focus in the lab involves immune evasion by Rhabdovirus that infect salmonid fish species and cause economic losses to the Rainbow Trout industry. The goals of the project include studying stress response pathways activated by various species of VHSV and flavobacteial pathogens to develop new vaccine candidates and antimicrobial peptides. The technology and knowledge gained from these studies will lead to eventually developing therapeutic options to improve Rainbow Trout production in the U.S.