Pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and RIG like helicase (RLH) receptors are involved in innate immune antiviral responses. Here we show that nucleotide-binding oligomerization domain 2 (NOD2) can also function as a cytoplasmic viral PRR by triggering activation of interferon regulatory factor-3 (IRF3) and production of interferon-β (IFN). Following recognition of viral ssRNA genome, NOD2 utilized the adaptor protein MAVS (mitochondrial antiviral signaling) to activate IRF3. NOD2-deficient mice failed to produce IFN efficiently and exhibited enhanced susceptibility to virus-induced pathogenesis. Thus, the function of NOD2 as a viral PRR highlights the important role of NOD2 in host antiviral defense mechanisms.

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although “controlled” inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the “first signal” constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two “second signals” are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K+) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-␣/␤-independent, innate antiviral response against RSV that was dependent on activation of NF-B. We demonstrated that NF-B inducing pro-inflammatory cytokines like tumor necrosis factor-␣ (TNF) confers potent antiviral function against RSV in an NF-B-dependent fashion, independent of interferon-␣/␤. During our efforts to study this pathway, we identified HBD2 (human ␤-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-B-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-B-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine ␤-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-B-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.Nonsegmented negative strand RNA viruses (superfamily Mononegaviridae) constitute highly pathogenic human viruses that cause high morbidity. Human respiratory syncytial virus (RSV), 2 a nonsegmented negative strand virus, is an important human lung tropic respiratory tract pathogen causing high morbidity and mortality among infants, children, and elderly by manifesting disease states, including pneumonia, and bronchiolitis (1, 2). To date, no effective vaccine or antiviral therapy exists for RSV. Therefore, elucidation of innate immune antiviral response induced by RSV holds significant potential for development of effective antiviral therapies in the near future.The innate immune antiviral response against viruses represents an important host defense mechanism (3). Innate immunity includes the first line of defense by the host to combat virus infection before an orchestrated adaptive immune response involving immune cell priming, and antibody production is launched. Two key molecules regulating the innate antiviral function are interferon regulatory factors (IRFs) and NF-B (4). These two transcription factors are activated either individually or together in infected cells, resulting in the expression and production of interferon-␣/␤ (IFN), which are potent antiviral cytokines (5, 6). IFN produced from infected cells binds to their...

Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.

Oncolytic virotherapy is an emerging bio-therapeutic platform for cancer treatment, which is based on selective infection/killing of cancer cells by viruses. Herein we identify the human respiratory syncytial virus (RSV) as an oncolytic virus. Using prostate cancer models, we show dramatic enhancement of RSV infectivity in vitro in the androgen-independent, highly metastatic PC-3 human prostate cancer cells compared to the non-tumorigenic RWPE-1 human prostate cells. The oncolytic efficiency of RSV was established in vivo using human prostate tumor xenografts in nude mice. Intra-tumoral and intra-peritoneal injections of RSV led to a significant regression of prostate tumors. Furthermore, enhanced viral burden in PC-3 cells led to selective destruction of PC-3 cancer cells in vitro and in xenograft tumors in vivo due to apoptosis triggered by the down-regulation of NF-κB activity (and the resulting loss of anti-apoptotic function of NF-κB) in RSV-infected PC-3 cells. The intrinsic (mitochondrial) pathway constitutes the major apoptotic pathway; however, the death-receptor-dependent extrinsic pathway, mediated by the paracrine/autocrine action of tumor necrosis factor-α produced from infected cells, also partly contributed to apoptosis. Thus, the oncolytic property of RSV can potentially be exploited to develop targeted therapeutics for the clinical management of prostate tumors.