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A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

Herpesviruses are ubiquitous DNA viruses that can establish latency and cause a range of mild to life-threatening diseases in humans. Upon infection, herpesviruses trigger the activation of several host antiviral defense programs that play critical roles in curbing virus replication and dissemination. Recent work from many groups has integrated our understanding of TRIM (tripartite motif) proteins, a specific group of E3 ligase enzymes, as pivotal orchestrators of mammalian antiviral immunity. In this review, we summarize recent advances in the modulation of innate immune signaling by TRIM proteins during herpesvirus infection, with a focus on the detection of herpes simplex virus type 1 (HSV-1, a prototype herpesvirus) by cGAS-STING, RIG-I-like receptors, and Toll-like receptors. We also review the latest progress in understanding the intricate relationship between herpesvirus replication and TRIM protein-regulated autophagy and apoptosis. Finally, we discuss the maneuvers used by HSV-1 and other herpesviruses to overcome TRIM protein-mediated virus restriction.

The cGAS-STING pathway, well-known to elicit interferon (IFN) responses, is also a key inducer of autophagy upon virus infection or other stimuli. Whereas the mediators for cGAS-induced IFN responses are well characterized, much less is known about how cGAS elicits autophagy. Here, we report that TRIM23, a unique TRIM protein harboring both ubiquitin E3 ligase and GTPase activity, is crucial for cGAS-STING-dependent antiviral autophagy. Genetic ablation of TRIM23 impairs autophagic control of HSV-1 infection. HSV-1 infection or cGAS-STING stimulation induces TBK1-mediated TRIM23 phosphorylation at S39, which triggers TRIM23 autoubiquitination and GTPase activity and ultimately elicits autophagy. Fibroblasts from a patient with herpes simplex encephalitis heterozygous for a dominant-negative, kinase-inactivating TBK1 mutation fail to activate autophagy by TRIM23 and cGAS-STING. Our results thus identify the cGAS-STING-TBK1-TRIM23 axis as a key autophagy defense pathway and may stimulate new therapeutic interventions for viral or inflammatory diseases. The cGAS-STING pathway senses cytosolic DNA to activate interferon responses, but has also been implicated in autophagy induction. Here the authors show that, during herpes simplex virus infection, cGAS-induced autophagy is mediated by TBK1-induced TRIM23 phosphorylation and downstream signaling events to assist in antiviral immunity.

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells. ISGylation plays a crucial role in the innate immune response and requires sequential activity of E1, E2, and E3 enzymes. Here, the authors present cyro-EM structures that reveal the molecular mechanisms underlying ISG15 activation by the E1 enzyme Uba7 and transfer to its cognate E2 enzyme UBE2L6.

The posttranslational modification (PTM) of innate immune sensor proteins by ubiquitin or ubiquitin-like proteins is crucial for regulating antiviral host responses. The cytoplasmic dsRNA receptor melanoma differentiation-associated protein 5 (MDA5) undergoes several PTMs including ISGylation within its first caspase activation and recruitment domain (CARD), which promotes MDA5 signaling. However, the relevance of MDA5 ISGylation for antiviral immunity in an infected organism has been elusive. Here, we generated knock-in mice ( ) in which the two major ISGylation sites, K23 and K43, in MDA5 were mutated. Primary cells derived from mice exhibited abrogated endogenous MDA5 ISGylation and an impaired ability of MDA5 to form oligomeric assemblies leading to blunted cytokine responses to MDA5 RNA-agonist stimulation or infection with encephalomyocarditis virus (EMCV) or West Nile virus. Phenocopying mice, the mice infected with EMCV displayed increased mortality, elevated viral titers, and an ablated induction of cytokines and chemokines compared to WT mice. Molecular studies identified human HERC5 (and its functional murine homolog HERC6) as the primary E3 ligases responsible for MDA5 ISGylation and activation. Taken together, these findings establish the importance of CARD ISGylation for MDA5-mediated RNA virus restriction, promoting potential avenues for immunomodulatory drug design for antiviral or anti-inflammatory applications.

Interferon-stimulated gene 15 (ISG15) regulates diverse cellular responses including antiviral immunity through its conjugation to proteins, a process known as ISGylation. Several pathogens, including SARS-CoV-2, subvert ISGylation by encoding deISGylating enzymes. However, the direct targets and physiological consequences of coronaviral deISGylation remain poorly defined. Here, we ablated the deISGylating activity of the SARS-CoV-2 papain-like protease (PLpro) and found that loss of deISGylation boosted innate immune activation, attenuated virus replication, and promoted viral clearance in human cells and in mice. Through untargeted metabolomics and ISGylome proteomics analyses, combined with functional studies, we discovered in molecular detail how the activities of key metabolic enzymes in glycolysis, the pentose phosphate pathway, and oxidative stress are controlled by PLpro deISGylation. These findings provide fundamental new insight into how reversible ISGylation regulates immunity and metabolic processes at the molecular level and highlight viral deISGylation as a major viral tactic for rewiring immunometabolism.