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Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Philipps-University Marburg

A randomized controlled study investigated the effects of therapeutic climbing in patients with chronic low back pain. Before and after 4 weeks of training, physical and mental well-being were measured by two questionnaires (36-Item Short Form Health Survey [SF-36]; Hannover Functional Ability Questionnaire for measuring back pain-related disability [FFbH-R]).

The effects of therapeutic climbing in patients with chronic low back pain: a randomized controlled study.

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.

Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages.

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Inclusion bodies are a site of ebolavirus replication.

Infections with human coronavirus EMC (HCoV-EMC) are associated with severe pneumonia. We demonstrate that HCoV-EMC resembles severe acute respiratory syndrome coronavirus (SARS-CoV) in productively infecting primary and continuous cells of the human airways and in preventing the induction of interferon regulatory factor 3 (IRF-3)-mediated antiviral alpha/beta interferon (IFN-α/β) responses. However, HCoV-EMC was markedly more sensitive to the antiviral state established by ectopic IFN. Thus, HCoV-EMC can utilize a broad range of human cell substrates and suppress IFN induction, but it does not reach the IFN resistance of SARS-CoV.

Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus.

Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5' (5'ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5'ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5'ppp dsRNA panhandle as a RIG-I activator.

Incoming RNA virus nucleocapsids containing a 5'-triphosphorylated genome activate RIG-I and antiviral signaling.

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.

IFIT2 is an effector protein of type I IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-α secretion and LPS-induced endotoxin shock.

Negative strand RNA viruses with a nonsegmented genome (ns-NSVs) or a segmented genome (s-NSVs) are an important source of human and animal diseases. Survival of the host from those infections is critically dependent on rapidly reacting innate immune responses. Two cytoplasmic RNA helicases, RIG-I and MDA5 (collectively termed RIG-I-like receptors, RLRs), are essential for recognizing virus-specific RNA structures to initiate a signalling cascade, resulting in the production of the antiviral type I interferons. Here, we will review the current knowledge and views on RLR agonists, RLR signalling, and the wide variety of countermeasures ns-NSVs and s-NSVs have evolved. Specific aspects include the consequences of genome segmentation for RLR activation and a discussion on the physiological ligands of RLRs.

RIG-I-like receptors and negative-strand RNA viruses: RLRly bird catches some worms.

The group of negative-stranded RNA viruses (NSVs) with a segmented genome comprises pathogens like influenza virus (eight segments), Rift Valley fever virus and Hantavirus (three segments), or Lassa virus (two segments). Partitioning the genome allows rapid evolution of new strains by reassortment. Each segment carries a short double-stranded (ds) 'panhandle' structure which serves as promoter. Similar dsRNA structures, however, represent the optimal ligand for RIG-I, a cytoplasmic pathogen sensor of the antiviral interferon response. Thus, segmenting a virus genome can entail an increased RIG-I sensitivity. Here, we outline the astonishingly diverse and efficient strategies by which segmented NSVs are compensating for the elevated number of RIG-I ligands in their genome.

Michaela Weber

Institute for Virology

Philipps-University Marburg

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE