Glenn F. Rall

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

Significance The interferons are small secreted proteins with powerful antiviral and cytotoxic properties. Here, we outline a signaling pathway activated by interferons that results in the precipitous necrotic death of susceptible cells. Interferon-induced necrosis proceeds via a novel, progressive mechanism that requires RNA transcription, as well as the sequential activity of three serine-threonine kinases: PKR, RIP1, and RIP3. This pronecrotic kinase cascade is normally held in check by FADD and caspases. As FADD can be disabled by phosphorylation during mitosis, our findings suggest the existence of a putative cell cycle-dependent checkpoint that licenses interferon-induced necrosis. Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1–RIP3 “necrosome” complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Immune-Mediated Protection from Measles Virus-Induced Central Nervous System Disease Is Noncytolytic and Gamma Interferon Dependent

ABSTRACT Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, virus infections that result in neuronal depletion, either by virus-mediated cell death or by induction of the cytolytic immune response, could cause permanent neurological impairment of the host. In a transgenic mouse model of measles virus (MV) infection of neurons, we have previously shown that the host T-cell response was required for resolution of infection in susceptible adult mice. In this report, we show that this protective response did not result in neuronal death, even during the peak of T-cell infiltration into the brain parenchyma. When susceptible mice were intercrossed with specific immune knockout mice, a critical role for gamma interferon (IFN-γ) was identified in protection against MV infection and CNS disease. Moreover, the addition of previously activated splenocytes or recombinant murine IFN-γ to MV-infected primary neurons resulted in the inhibition of viral replication in the absence of neuronal death. Together, these data support the hypothesis that the host immune response can promote viral clearance without concomitant neuronal loss, a process that appears to be mediated by cytokines.

Measles Virus Spread between Neurons Requires Cell Contact but Not CD46 Expression, Syncytium Formation, or Extracellular Virus Production

ABSTRACT In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46+ mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46+ neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46+ and CD46− neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.

Making It to the Synapse : Measles Virus Spread in and Among Neurons

Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understanding of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environment of the neuron affects MV replication, spread, and, ultimately, neuropathogenesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression.

Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.

Model Systems: Transgenic mouse models for measles pathogenesis

Studies of the diseases caused by measles virus (MV) in humans have been restricted owing to the lack of suitable animal models. The discovery of cellular receptors for MV entry has facilitated the development of transgenic mice that are susceptible to MV infection, and that mimic certain aspects of the central nervous system diseases and immunosuppression that can occur in infected humans. Moreover, such mouse models have allowed a clearer understanding of the contributions of the innate and adaptive immune response following infection, and will no doubt be important tools in the future for the development of new antiviral and vaccine reagents.

Neuronal Survival Strategies in the Face of RNA Viral Infection

Abstract Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, viral infections that result in neuronal depletion, either by viral lysis or by induction of the cytolytic immune response, would likely lead to profound neurologic impairment. However, many viral infections that result in tissue destruction elsewhere in the host produce few overt symptoms in the CNS, despite readily detectable virus expression. This observation has lead to the speculation that neurons possess strategies to limit the replication and spread of otherwise cytopathic viruses. These strategies either favor the clearance of virus in the absence of appreciable neuronal loss or promote the establishment of noncytolytic persistent infections. This review discusses some of these strategies, with an emphasis on how such survival techniques lessen the potential for CNS neuropathology

Transduction of Terminally Differentiated Neurons by Avian Sarcoma Virus

ABSTRACT Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.

Lymphocytic choriomeningitis virus-induced mortality in mice is triggered by edema and brain herniation.

ABSTRACT Although much is known about lymphocytic choriomeningitis virus (LCMV) infection and the subsequent immune response in its natural murine host, some crucial aspects of LCMV-mediated pathogenesis remain undefined, including the underlying basis of the characteristic central nervous system disease that occurs following intracerebral (i.c.) challenge. We show that the classic seizures and paresis that occur following i.c. infection of adult, immunocompetent mice with LCMV are accompanied by anatomical and histological changes that are consistent with brain herniation, likely of the uncal subtype, as a causative basis for disease and precipitous death. Both by water weight determinations and by magnetic resonance imaging of infected brain tissues, edema was detected only at the terminal stages of disease, likely caused by the leakage of cerebrospinal fluid from the ventricles into the parenchyma. Furthermore, death was accompanied by unilateral pupillary dilation, which is indicative of uncal herniation. While immunohistochemical analysis revealed periventricular inflammation and a loss of integrity of the blood-brain barrier (BBB), these events preceded seizures by 2 to 3 days. Moreover, surviving perforin knockout mice showed barrier permeability equivalent to that of moribund, immunocompetent mice; thus, BBB damage does not appear to be the basis of LCMV-induced neuropathogenesis. Importantly, brain herniation can occur in humans as a consequence of injuries that would be predicted to increase intracranial pressure, including inflammation, head trauma, and brain tumors. Thus, a mechanistic dissection of the basis of LCMV neuropathogenesis may be informative for the development of interventive therapies to prevent this typically fatal human condition.

CNS Neurons: The Basis and Benefits of Low Class I Major Histocompatibility Complex Expression

The host immune response is generally thought to consist of cells with “professional” immunologic functions, such as B and T cells, macrophages, and natural killer (NK) cells. However, differentiated cells which do not normally participate in immune surveillance may be recruited to serve an integral function in the immune-mediated elimination of foreign intracellular pathogens such as viruses. As discussed elsewhere in this volume, most cells have the ability to present immunogenic, “non-self” peptides (called epitopes) in association with “self&” class I major histocompatibility complex (MHC) molecules. This cell surface complex is engaged by the T cell receptor (TCR) of cytotoxic T lymphocytes (CTL). Appropriate MHC-epitope-TCR interaction leads to the CTL-mediated lysis of the epitope-expressing target cell via the perforation of the plasma membrane, introduction of CTL-derived proteolytic enzymes (granzymes) into the target cell cytosol, and eventual cell death, presumably via apoptosis.