Identification of Effective siRNA Blocking the Expression of SARS Viral Envelope E and RDRP Genes

Abstract

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.

siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA

“…siRNA targeting the E, M and N proteins , leader sequence, TRS, 3 -UTR or the S gene Qin et al, 2004;Zhang et al, 2004) could effectively inhibit the expression of these targets. Plasmid-derived siRNA targeting to RDRP (Meng et al, 2006;Wang et al, 2004), the leader sequence (B.J. T.…”

Polymerases of Coronaviruses

“…The results showed that all anti-SARS siRNA inhibited viral gene expression. This finding suggested a novel approach for the inhibition of production of viral proteins leading to treatment of SARS (Meng et al, 2006;Bitko and Barik, 2007).…”

“…There are two approaches to produce siRNAs in mammalian cells, the first is under the control of specific promoters to form doublestranded siRNA, and the second is through a hairpin structure where siRNAs are expressed as hairpin structures and subsequently processed to siRNA intracellularly. Meng et al employed both the approaches to produce several siRNA molecules to test against SARS-CoV RdRP and envelope E protein (Meng et al, 2006). In this study, a cell-based assay was developed for screening siRNA to inhibit the expression of two genes that encode envelope E protein and RdRP of SARS-CoV.…”

“…Reverse transcription was used to examine the expression levels of envelope E genes and RdRP, followed by quantitative real-time polymerase chain reaction (PCR). Approximately 90% of envelope E gene expression was reduced by both siRNA expression cassettes for envelope E, while two of the siRNA expression cassettes for RdRP effectively blocked the expression of these genes (Kamath and Ahringer, 2003;Boutros et al, 2004;Paddison et al, 2004;Meng et al, 2006).…”

“…These siRNA molecules may be useful for further studies to test the function of these genes in SARS virus replication and assembly. Furthermore, these agents could potentially be developed into therapeutic options for SARS treatment (Meng et al, 2006).…”