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.

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 (...

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