Annie M. Bruns

Activation of RIG-I-like receptor signal transduction

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed.

ATP Hydrolysis Enhances RNA Recognition and Antiviral Signal Transduction by the Innate Immune Sensor, Laboratory of Genetics and Physiology 2 (LGP2)

Background: Laboratory of genetics and physiology 2 (LGP2) is a cytoplasmic RNA receptor required for innate antiviral signaling. Results: LGP2 uses ATP hydrolysis to diversify RNA recognition and enhance antiviral signaling. Conclusion: LGP2 mediates antiviral responses by ATP-enhanced RNA recognition. Significance: This study reveals a novel property of LGP2 providing a mechanistic basis for its positive role in antiviral signaling. Laboratory of genetics and physiology 2 (LGP2) is a member of the RIG-I-like receptor family of cytoplasmic pattern recognition receptors that detect molecular signatures of virus infection and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2 is essential for antiviral signaling, but it has been unclear how the enzymatic properties of LGP2 regulate its biological response. Quantitative analysis of the dsRNA binding and enzymatic activities of LGP2 revealed high dsRNA-independent ATP hydrolysis activity. Biochemical assays and single-molecule analysis of LGP2 and mutant variants that dissociate basal from dsRNA-stimulated ATP hydrolysis demonstrate that LGP2 utilizes basal ATP hydrolysis to enhance and diversify its RNA recognition capacity, enabling the protein to associate with intrinsically poor substrates. This property is required for LGP2 to synergize with another RIG-I-like receptor, MDA5, to potentiate IFNβ transcription in vivo during infection with encephalomyocarditis virus or transfection with poly(I:C). These results demonstrate previously unrecognized properties of LGP2 ATP hydrolysis and RNA interaction and provide a mechanistic basis for a positive regulatory role for LGP2 in antiviral signaling.

Radiation-induced loss of cell surface CD47 enhances immune-mediated clearance of human papillomavirus-positive cancer

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(−) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune‐mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation‐induced change in tumor surface protein expression that is critical for immune‐mediated clearance. Radiation therapy decreases surface expression of CD47, a self‐marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose‐dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune‐mediated tumor clearance in vivo. These findings help define an important mechanism of radiation‐related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

MDA5 and LGP2: Accomplices and Antagonists of Antiviral Signal Transduction

ABSTRACT Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction.

LGP2 synergy with MDA5 in RLR-mediated RNA recognition and antiviral signaling

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response.

Antiviral RNA Recognition and Assembly by RLR Family Innate Immune Sensors

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors. One important group of cytosolic RNA sensors known as the RIG-I-like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation.

Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antiviral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production and cytidine-to-uridine-driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3′-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires the specific interaction of A3G with reverse transcriptase itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between reverse transcriptase, A3G and cellular DNA repair machinery, and identify the suppression of HIV-1 reverse transcriptase by a directly interacting host protein as a new cell-mediated antiviral mechanism.APOBEC3G is shown to induce a potent, non-site-specific interference with reverse transcription through direct interaction with HIV-1 reverse transcriptase, and host DNA repair machinery is shown to cleave HIV-1 cDNA.

Radiation-induced loss of cell surface CD47 enhances immune- mediated clearance of HPV+ cancer

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(−) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune‐mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation‐induced change in tumor surface protein expression that is critical for immune‐mediated clearance. Radiation therapy decreases surface expression of CD47, a self‐marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose‐dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune‐mediated tumor clearance in vivo. These findings help define an important mechanism of radiation‐related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG‐I and MDA5 stimulate a mitochondria‐localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA. LGP2 associates with the C‐terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C‐terminal domain, and LGP2 can regulate TRAF‐mediated signaling pathways in trans, including IL‐1β, TNFα, and cGAMP. These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response.

Radiation-induced loss of cell surface CD47 enhances immune-mediated clearance of human papillomavirus-positive cancer: Radiation-decreased CD47 improves tumor clearance

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(−) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune‐mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation‐induced change in tumor surface protein expression that is critical for immune‐mediated clearance. Radiation therapy decreases surface expression of CD47, a self‐marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose‐dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune‐mediated tumor clearance in vivo. These findings help define an important mechanism of radiation‐related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.