siRNA-Mediated Simultaneous Regulation of the Cellular Innate Immune Response and Human Respiratory Syncytial Virus Replication

Abstract

Human respiratory syncytial virus (HRSV) infection is a common cause of severe lower respiratory tract diseases such as bronchiolitis and pneumonia. Both virus replication and the associated inflammatory immune response are believed to be behind these pathologies. So far, no vaccine or effective treatment is available for this viral infection. With the aim of finding new strategies to counteract HRSV replication and modulate the immune response, specific small interfering RNAs (siRNAs) were generated targeting the mRNA coding for the viral fusion (F) protein or nucleoprotein (N), or for two proteins involved in intracellular immune signaling, which are named tripartite motif-containing protein 25 (TRIM25) and retinoic acid-inducible gene-I (RIG-I). Furthermore, two additional bispecific siRNAs were designed that silenced F and TRIM25 (TRIM25/HRSV-F) or N and RIG-I (RIG-I/HRSV-N) simultaneously. All siRNAs targeting N or F, but not those silencing TRIM25 or RIG-I alone, significantly reduced viral titers. However, while siRNAs targeting F inhibited only the expression of the F mRNA and protein, the siRNAs targeting N led to a general inhibition of viral mRNA and protein expression. The N-targeting siRNAs also induced a drastic decrease in the expression of genes of the innate immune response. These results show that both virus replication and the early innate immune response can be regulated by targeting distinct viral products with siRNAs, which may be related to the different role of each protein in the life cycle of the virus.

Downregulation of A20 Expression Increases the Immune Response and Apoptosis and Reduces Virus Production in Cells Infected by the Human Respiratory Syncytial Virus

“…Second, downregulation of A20, TAX1BP1 and ABIN1 decreased virus production at later times post-infection, and this may have contributed to reducing the intracellular immune response at this time. Inhibition of HRSV replication has been shown to decrease the inflammatory/antiviral response in infected cells [67].…”

Metabolic changes during respiratory syncytial virus infection of epithelial cells

“…Epithelial cells and HRSV were cultured as previously described [30]. In brief, Human lung carcinoma cells (A549, ATCC1 CCL-185) and human carcinoma HeLa-derived cells (HEp-2, ATCC1 CCL-23) were grown in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, Utah, USA) complemented with 10% fetal bovine serum (FBS, Biological Industries, Beit HaEmerk, Israel), 4 mM L-Glutamine (HyClone), 100 U/ml penicillin (Lonza, Verviers, Belgium) and 100 μg/ml streptomycin (HyClone) (DMEM10).…”

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

“…During the search of effective treatment for CMV, idea of controlling the disease by means of inhibiting virus replication and gene expression by combinations of two and more siRNAs had been explored in this study. Combination therapies have been tested before targeting other viral infections such as influenza, coronavirus, respiratory syncytial virus, hepatitis B&C, vaccinia virus [ 23 , 25 , 43 – 46 ]. It was hypothesized that targeting two different genes simultaneously could silence the expression of crucial viral genes thus; act as an antiviral therapy against CMV.…”