Inhibition Of Viral Gene Expression And Replication By RNA Interference

The replication of a growing number of human pathogenic viruses in cell culture was shown to be inhibited by RNAi, including poliovirus , flock house virus (FHV) , Rous sarcoma virus, dengue virus, poliovirus, HCV replicons, influenza virus, hepatitis B virus (HBV) , HPV, recently it was reported that RNAi can also induce transcriptional silencing of SARS coronvirus. In most above studies, synthetic 21-nucleotide double-stranded siRNAs were applied. However, vector based RNAi techniques were used more frequently in recent studies. Each vector expresses unique siRNA that can degrade specific target.In this study, we applied a different approach by designing a pair of 64nt primers that contain a specific 19nt target sequence from HBV genome to create recombinant pSilencer-U6 plasmid. Primers were annealed and cloned into BamHI-HindIII sites of the pSilencer2.1-U6 vector. In order to construct a useful tool to choose the most effective siRNA molecules, we created a quick screening vector plucF by fusing the targeted sequence and the reporter luciferase gene together to produce recombinant plucF plasmid, which could express HBS-luciferase or HBx-luciferase fusion mRNAs. Therefore, we can initially select the suitable siRNA duplexes rapidly by simply analyzing the activities of luciferase. By using this approach, we have identified 2 siRNA molecules (HBssiRNAl and HBssiRNA2) having significant impact on the HBs-luciferase fusion gene expression and 1 siRNA duplex (HBxsiRNA2) having effects on the expression of HBx-luciferase fusion gene. This provides a quick approach to select effective siRNA in the study of gene expression and function analysis.To further study the effects of selected RNAi molecules on HBV gene expression and viral replication in a cell culture models, we used a derivative of the human HepG2 hepatoma cell line, HepG2 2.2.15, which has been stably transformed with several copies of the HBV genome and used as an in vitro model for HBV replication. The effects of dual siRNA system on HBV gene expression and viral replication were studied thoroughly by the analyzing the levels of viral protein production through enzyme-linked immunosorbent assay and the levels of viral RNA expression by semi-quantitatedRT-PCR analysis. All results indicated that HBssiRNAi, HBssiRNA2, HBxsiRNA2 had significant reduction effects on viral mRNA expression, and viral protein production.A number of clinic studies have been shown that human immunodeficiency virus (HIV ) is often acquired in individuals already infected with hepatitis B virus (HBV ) as a result of shared routes of transmission. Antiretroviral therapy for HIV, and associated immune reconstitution, may result in immune-mediated liver damage as HBV-infected hepatocytes are targeted. Since currently available options for the treatment of co-infection of HIV and HBV are limited, there is an essential need for the development of effective therapies against these viral infections. We have established previously a dual small interfering RNA (siRNA) expression system, which provides the possibility to control disease of viral co-infection. So we here extended our study by using this system to produce simultaneously two siRNA duplexes that targeted the S gene of HBV and the gpl20 gene of HIV-1, respectively. Our results demonstrated that the two siRNA molecules generated from this system could simultaneously inhibit the expression of HBs and gpl20 respectively. In addition, dual siRNA molecules decreased significantly the production of HBs and p24 proteins and inhibited the replication of HBV inHepG2.2.15 cells and HIV in HEK293T cells simultaneously. Based on these results, we believed that this dual siRNA generation system not only provides a novel approach for the study of functions of multiple genes simultaneously, but also provide a potential application in the treatment and prevention of HIV and HBV co-infection.