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.

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), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKK, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo-as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKK reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. Human respiratory syncytial virus (RSV) is a member of thePneumovirus genus in the Paramyxoviridae family (9, 25). RSV replicates primarily in the respiratory epithelium and is a leading cause of respiratory disease and asthma in the very young and the elderly (41, 42). The nonsegmented negative-strand RNA genome of RSV encodes 10 genes, of which the two most promoterproximal and abundantly transcribed genes code for nonstructural proteins 1 and 2 (NS1 and NS2), which are small proteins, 139 and 124 amino acid residues long, respectively (9, 25). The NS proteins circumvent the host innate immune system by preventing the induction of type I interferons (IFNs) as well as IFN-induced antiviral responses (6,12,20,22,23,29,(33)(34)(35)(36)39), thus allowing a more robust replication of the virus, leading to the severe respiratory disease that characterizes RSV infection.The IFN pathways of the cell can be divided into two functional ones, the IFN induction pathway, in which the cells produce IFN, and the IFN response pathway, in which the cells respond to exogenous IFN. One of the most proximal steps in the IFN induction pathway is the activation of the cytoplasmic RNA sensors of the RIG-I family (27,37,43,44). This triggers a cascade of signaling events whereby the CARD sequence of RIG-I interacts with and activates the CARD-like domaincontaining mitochondrial protein, MAVS (7, 18). Activated MAVS then activates TRAF3 (7, 31), which in turn activates two downstream kinases, IKKε and TBK1. The latter are Ser/ Thr kinases that phosphorylate the C-terminal domain of interferon regulatory factor 3 (IRF3) and IRF7, leading to...

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-B and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN-and EGFR-independent pathway.The respiratory syncytial virus (RSV), a member of the Pneumovirus genus within the family Paramyxoviridae, is a ubiquitous cause of severe respiratory infection with a worldwide and seasonal distribution (26). It is the most common cause of lower respiratory tract infection in infants and senior citizens and is life-threatening in immunocompromised individuals such as premature babies, organ recipients, and AIDS patients. Large-scale surveillance studies have estimated that tens of millions of people in the United States alone suffer from RSV infection every winter. Despite intense research for more than two decades, there is no reliable treatment or preventive medicine against RSV.The RSV genome is a 15-kb-long, single-stranded, negativesense RNA, transcribed and replicated by the virally encoded RNA-dependent RNA polymerase (RdRP), minimally composed of the large protein (L) and the phosphoprotein (P). The overall strategy of RSV gene expression is common to all members of the negative-strand RNA virus superfamily (2, 28). The initial rounds of transcription, known as "primary" transcription, are carried out by the RdRP activity associated with the incoming viral genome. The transcribed mRNAs are translated into de novo viral proteins including more RdRP, which boosts new rounds of viral gene expression. Perhaps the most unique feature that distinguishes the Pneumovirus genus from the rest of the Paramyxoviridae family is the presence of two nonstructural (...

MicroRNAs (miRNAs) are endogenous noncoding RNAs that down-regulate gene expression by promoting cleavage or translational arrest of target mRNAs. While most miRNAs are transcribed from their own dedicated genes, some map to introns of 'host' transcripts, the biological significance of which remains unknown. Here, we show that prostate cells are naturally devoid of EGF-like domain 7 (Egfl7) transcripts and hence also deficient in a miRNA, miR-126*, generated from splicing and processing of its ninth intron. Use of recombinant and synthetic miRNAs or a specific antagomir established a role of miR-126* in silencing prostein in non-endothelial cells. We mapped two miR-126*-binding sites in the 3′UTR of the prostein mRNA required for translational repression. Transfection of synthetic miR-126* into prostate cancer LNCaP cells strongly reduced the translation of prostein. Interestingly, loss of prostein correlated with reduction of LNCaP cell migration and invasion. Thus, the robust expression of prostein protein in the prostate cells results from a combination of transcriptional activation of the prostein gene and absence of intronic miRNA-126* due to the prostate-specific repression of the Egfl7 gene. We conclude that intronic miRNAs from tissue-specific transcripts, or their natural absence, make cardinal contributions to cellular gene expression and phenotype. These findings also open the door to tissue-specific miRNA therapy.