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.

Infection of the lung epithelial cell line A549 by respiratory syncytial virus (RSV) resulted in the elevated synthesis of multiple cellular cytokines, including a number of interleukins (ILs). Detailed studies of IL-11 induction revealed that it required infection by viable virus and involved a net increase in the steady state level of IL-11 mRNA. Nuclear run-on assays showed a direct effect of RSV on IL-11 gene transcription. Mutational analysis of the IL-11 promoter fused to a reporter luciferase gene demonstrated the requirement of a region 720 nucleotides upstream of the mRNA start site in the transcriptional induction of IL-11 by RSV. Two nearly identical 10-nucleotide-long sequences GGGGTCTCCC and GGGTCTCCCC in this region resembled the NF-kappa B consensus motif. Mutation of either sequence greatly reduced RSV-mediated induction of IL-11 promoter activity. NF-kappa B sites in IL-1 alpha, IL-6, and IL-8 promoters were also required for RSV-mediated induction of transcription of these promoters. Immunological studies and use of reporter gene constructs provided direct evidence for the activation and nuclear translocation of NF-kappa B by RSV. Sodium salicylate and aspirin, inhibitors of NF-kappa B activation, abolished transcriptional induction of all these cytokines by RSV. Together, these studies demonstrated an essential role of NF-kappa B in RSV-mediated transcription of multiple cytokines genes and suggested a possible use of salicylates in managing airway inflammation and viral pathogenesis during RSV infection.

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 (...

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.

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.