Farhad Imani

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

Short interfering RNAs (siRNAs) are double-stranded RNAs of ≈21–25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5′-phosphate/3′-hydroxyl ends and a 2-base 3′ overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.

Cell Cycle Arrest by Transforming Growth Factor β1 Enhances Replication of Respiratory Syncytial Virus in Lung Epithelial Cells

ABSTRACT Respiratory syncytial virus (RSV) is a common respiratory viral infection in children which is associated with immune dysregulation and subsequent induction and exacerbations of asthma. We recently reported that treatment of primary human epithelial cells (PHBE cells) with transforming growth factor β (TGF-β) enhanced RSV replication. Here, we report that the enhancement of RSV replication is mediated by induction of cell cycle arrest. These data were confirmed by using pharmacologic inhibitors of cell cycle progression, which significantly enhanced RSV replication. Our data also showed that RSV infection alone resulted in cell cycle arrest in A549 and PHBE cells. Interestingly, our data showed that RSV infection induced the expression of TGF-β in epithelial cells. Blocking of TGF-β with anti-TGF-β antibody or use of a specific TGF-β receptor signaling inhibitor resulted in rescue of the RSV-induced cell cycle arrest, suggesting an autocrine mechanism. Collectively, our data demonstrate that RSV regulates the cell cycle through TGF-β in order to enhance its replication. These findings identify a novel pathway for upregulation of virus replication and suggest a plausible mechanism for association of RSV with immune dysregulation and asthma.

Transforming Growth Factor β Enhances Respiratory Syncytial Virus Replication and Tumor Necrosis Factor Alpha Induction in Human Epithelial Cells

ABSTRACT Asthma is characterized as a chronic inflammatory disease associated with significant tissue remodeling. Patients with asthma are more susceptible to virus-induced exacerbation, which subsequently can lead to increased rates of hospitalization and mortality. While the most common cause of asthma-related deaths is respiratory viral infections, the underlying factors in the lung environment which render asthmatic subjects more susceptible to viral exacerbation are not yet identified. Since transforming growth factor β (TGF-β) is a critical cytokine for lung tissue remodeling and asthma phenotype, we have focused on the effects of TGF-β on viral replication and virus-induced inflammation. Treatment of human epithelial cells with TGF-β increased respiratory syncytial virus (RSV) replication by approximately fourfold. Tumor necrosis factor alpha (TNF-α) mRNA and protein expression were also significantly increased above levels with RSV infection alone. The increase in RSV replication and TNF-α expression after TGF-β treatment was concomitant with an increase in virus-induced p38 mitogen-activated protein kinase activation. Our data reveal a novel effect for TGF-β on RSV replication and provide a potential mechanism for the exaggerated inflammatory response observed in asthmatic subjects during respiratory viral infections.