Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are two respiratory pathogens of paramount medical significance that exert high mortality. At present, there is no reliable vaccine or antiviral drug against either virus. Using an RNA interference (RNAi) approach, we show that individual as well as joint infection by RSV and PIV can be specifically prevented and inhibited by short interfering RNAs (siRNAs), instilled intranasally in the mouse, with or without transfection reagents. The degree of protection matched the antiviral activity of the siRNA in cell culture, allowing an avenue for quick screening of an efficacious siRNA. When targeting both viruses in a joint infection, excess of one siRNA moderated the inhibitory effect of the other, suggesting competition for the RNAi machinery. Our results suggest that, if properly designed, low dosages of inhaled siRNA might offer a fast, potent and easily administrable antiviral regimen against respiratory viral diseases in humans.

SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Immunophilins are chaperones that may also exhibit peptidylprolyl isomerase (PPIase) activity. This review summarizes our knowledge of the two largest families of immunophilins, namely cyclophilin and FK506-binding protein, and a novel chimeric dual-family immunophilin, named FK506- and cyclosporin-binding protein (FCBP). The larger members of each family are modular in nature, consisting of multiple PPIase and/or protein-protein interaction domains. Despite the apparent difference in their sequence and three-dimensional structure, the three families encode similar enzymatic and biological functions. Recent studies have revealed that many immunophilins possess a chaperone function independent of PPIase activity. Knockout animal studies have confirmed multiple essential roles of immunophilins in physiology and development. An immunophilin is indeed a natural 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper folding and assembly.

Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKK, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKK proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKK levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKK levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKK and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.Respiratory syncytial virus (RSV) is by far the most significant agent of pediatric respiratory disease for which no reliable antiviral or vaccine yet exists (45, 59). The lukewarm success of traditional active-immunization-based strategies has drawn focus to cellular innate immunity, which acts as a broad antiviral defense. A major arm of innate antiviral immunity is the type I IFN family, represented by alpha interferon (IFN-␣) and 46,58). In counterdefense, however, members of the Paramyxoviridae family have evolved accessory gene products that neutralize or inhibit various steps of the IFN pathway, thus permitting robust virus growth (18,27,57,71). It is now appreciated that a better understanding of the viral IFN suppression mechanism(s) is essential for the prudent design of attenuated vaccine strains and better overall therapy.RSV encodes unique anti-IFN genes not found in any other member virus of this family. While other viruses generate IFNsuppressive proteins, mainly the V protein (1,12,18,19,21,22,27,33,50,51,57), from alternative translational reading frames in the P gene through "RNA editing," the P gene of RSV codes for the P protein only. Instead, the RSV genome contains two promoter-proximal genes that code for nonstructural (NS) proteins, NS1 and NS2, which are so named because they are synthesized in RSV-infected cells but are not packaged in the mature virion structure. The predicted primary structures of the NS proteins do not share any sig...

Human respiratory syncytial virus (RSV) inhibits type I interferon-induced gene expression by decreasing expression of signal transducer and activator of transcription (Stat)2. To identify the RSV protein that mediates effects on Stat2, airway epithelial cells were infected with vaccinia virus vectors that express single RSV proteins. Expression of RSV nonstructural (NS)2 protein alone was sufficient to decrease Stat2 levels. Furthermore, decreasing RSV NS2 levels using RNA interference in respiratory epithelial cells inhibited the RSV-mediated decrease in Stat2 expression. Airway epithelial cells were also infected with equivalent inoculums of RSV without or with single gene deletions of NS1 or NS2. RSV infection without NS2 expression did not result in decreased Stat2 levels or loss of type I interferon-dependent signaling, indicating that NS2 expression is necessary for RSV effects on Stat2. Taken together, our results indicate that NS2 regulates Stat2 levels during RSV infection, thereby modulating viral effects on interferon-dependent gene expression.