Attenuation of SARS coronavirus by a short hairpin RNA expression plasmid targeting RNA-dependent RNA polymerase

Abstract

Severe acute respiratory syndrome (SARS) is a highly contagious and sometimes a lethal disease, which spread over five continents in 2002-2003. Laboratory analysis showed that the etiologic agent for SARS is a new type of coronavirus. Currently, there is no specific treatment for this disease. RNA interference (RNAi) is a recently discovered antiviral mechanism in plant and animal cells that induces a specific degradation of double-stranded RNA. Here, we provide evidences that RNAi targeting at coronavirus RNA-dependent RNA polymerase (RDRP) using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Moreover, this construct significantly reduced the plaque formation of SARS coronaviruses in Vero-E6 cells. The data may suggest a new approach for treatment of SARS patients.

Interferon-Inducible Ubiquitin E2, Ubc8, Is a Conjugating Enzyme for Protein ISGylation

“…The PCR product was digested with BamHI and EcoRI and ligated to MSCV-PGKpuro (BD Biosciences Clontech) digested with BglII and EcoRI. UbcH8 knockdown experiments were performed by transiently expressing an shRNA construct for cells (29). To test the efficiency of UbcH8 shRNA in cotransfection experiments, 293T cells were transfected with MSCV-UbcH8 shRNA, pFlagCMV2-UbcH8, pCAGGS-mISG15, and pCAGGS-HA-hUBE1L.…”

dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus

“…In other words, BmCPV-RDRP functions as both replicase and transcriptase. Molecular therapy of some human viral disease by dsRNA interference are targeted to RDPD, including RNA viruses such as SARS virus (Lu et al, 2004) and hepatitis virus (McCaffrey et al, 2003). Most studies on anti-virus transgenic animals and plants are also targeted to RDRP genes (Ahlquist, 2002).…”

Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene

“…Viral RdRp plays an essential role in viral RNA replication. Many researchers have shown that siRNA/shRNA targeting the RdRp coding gene could efficiently inhibit viral replication; moreover, according to their reports, viral inhibition triggered by siRNA/shRNA targeting the RdRp gene seems more efficient compared to other genes within the same genome (Ge et al, 2003;Lu et al, 2004;Wang et al, 2004). Therefore, the RdRp gene of TGEV, which is highly conserved among different strains, was employed as an RNAi target in this study, and our results demonstrated that two shRNAs generated from it both blocked viral replication with very high efficiency.…”