Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.

The innate immune system is a vital part of the body's defences against viral pathogens. The proteins retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) function as cytoplasmic pattern recognition receptors that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualisation of dsRNA and MDA5, which provides unique evidence of the relationship between viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.

Human respiratory syncytial virus viroporin SH: a viral recognition pathway used by the host to signal inflammasome activation.

BACKGROUND: Respiratory syncytial virus (RSV) remains the leading cause of serious viral bronchiolitis and pneumonia in infants and young children throughout the world. The burden of disease is significant, with 70% of all infants being infected with RSV within the first year of their life. 40% of those children discharged from hospital have recurrent, repeated respiratory symptoms and wheezing for at least 10 years. The infection is also an important illness in the elderly and immunocompromised individuals. Ongoing symptoms relate to continued lung inflammation. One cytokine that is associated with RSV infection is IL-1ß, but the mechanism of activation remain unclear. OBJECTIVES: In the current study, we set out to decipher the molecular mechanisms of RSV-induced inflammasome activation. METHODS AND RESULTS: Using deletion mutants of the virus and measuring IL-1ß secretion, as well as caspase 1 expression via western blotting, we demonstrate that the NLRP3 inflammasome is activated through the small hydrophobic (SH) RSV viroporin which induces membrane permeability to ions or small molecules. Confocal microscopy revealed that during virus infection, SH seems to accumulate within lipid rafts in the Golgi compartments. CONCLUSIONS: Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation.

Cell surface molecular chaperones as endogenous modulators of the innate immune response.

Mammalian responses to bacterial products can lead to an uncontrolled inflammatory response that can be deadly for the host. It has been shown that the innate immune system employs at least three cell surface receptors, TLR4, CD14 and MD2, in order to recognize bacterial products. We have previously shown that heat shock proteins (HSPs) are also involved in the innate immune recognition. HSPs are a family of highly conserved proteins that act as molecular chaperones and assist in proper folding, assembly and intracellular trafficking of proteins. How HSPs reach the cell surface and how they are involved in the innate immune response still remain unclear. In the present study we investigated their association with the TLR4/CD14/MD2 complex in response to bacterial products and provide evidence that the Hsp70 and Hsp90 associate with TLR4 on the cell surface in response to stimulation by bacterial products. These associations seem to take place within lipid rafts. The addition of exogenous recombinant Hsp70 to cells in vitro results in a dose-responsive inhibition of the inflammatory signal cascade and cytokine production. Our studies reveal that HSPs may play an important role as endogenous regulators of the innate immune response.

Human rhinovirus recognition in non-immune cells is mediated by Toll-like receptors and MDA-5, which trigger a synergetic pro-inflammatory immune response.

The early detection of invading viruses by the host depends on their identification by pathogen sensors. These include Toll-like receptors (TLRs) as well as cytoplasmic RNA helicases such as retinoic acid inducible protein I (RIG-I) and melanoma differentiation associated gene 5 (MDA-5). These pathogen sensors recognize specific molecular patterns found in viruses and trigger inflammatory and antiviral responses that result in the eradication of invading pathogens. In this study we investigated the specific recognition of Human rhinovirus 6 (HRV6) the common cold pathogen by the innate immune response in lung epithelial cells. Our experiments established that in the first stages on infection the TLRs play a crucial role in HRV recognition and that different constituents of HRV6 are recognized by different TLRs, while upon viral replication and generation of dsRNA the type I IFN inflammatory response is mediated by MDA-5. The HRV6 capsid is recognized via TLR2, whereas upon HRV6 ssRNA internalization the virus genome is recognized by TLR7 and TLR8. Upon generation of dsRNA the type I IFN response is mediated by MDA-5. The combined recognition by different TLRs and MDA5 and their upregulation concurs with the huge inflammatory response seen in the common cold caused by human rhinoviruses.

TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1.

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.

Human cardiac inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent.

The group B coxsackieviruses are single-stranded RNA viruses that have been implicated in viral myocarditis. Viral infection of the myocardium, as well as the associated inflammatory response are important determinants of the virus-associated myocardial damage. Although these viruses are known as cytopathic viruses that cause death of the host cell, their viral RNA has been shown to persist in cardiac muscle contributing to a chronic inflammatory cardiomyopathy. Thus, it is essential that we understand the mechanism by which Coxasckie B viruses (CBVs) trigger this inflammatory response. In this study we investigated the involvement of Toll-like receptors (TLRs) in the recognition of CBV virions as well as CBV single-stranded RNA. Here we report that the CBV-induced inflammatory response is mediated through TLR8 and to a lesser extent through TLR7.