A Study Of The Effects Of ICP27-specific SiRNA On Inhibiting The Replication Of HSV-1

Herpes simplex virus encephalitis (HSE) is one of the most common types of diverging virus encephalitis. Its symptome is serious, development is fast, and fatality rate is high. The anti-HSV-1 medicine can't complete suppression and clean the latent virus. It also has drug resistance.95 percent of HSE is caused by herpes simplex virus type 1 (HSV-1), which is a kind of double-stranded DNA virus. According to the time point of expression after infection, HSV-1 genome is grouped into immediate early (IE), early (E) and late (L) genes. HSV-1 infected-cell protein 27 (ICP27) is 63KDa immediate-early regulatory phosphoprotein, which is produced by a27 gene. The N-terminus is rich of acidic amino acid, and the C-terminus is rich of cysteine. ICP27 is a key activator during the replication of HSV-1, simulating the expression and synthesis of E genes and induces the expressing of L genes. Moreover, ICP27 inhabits host cell protein synthesis by mediating the metabolism of the mRNA of host cell.RNAi (RNA interference) is a gene-silencing mechanism based on sequence-specific targeting and post-transcriptional mRNA degradation, which is induced by double-stranded RNA. As a new efficient and specific tool for gene knockdown, RNAi technology has developed rapidly in recent years and has a wide application prospect in the gene therapy of virus.The study established the recombination cell lines expressing the ICP27 sequence-specific siRNAs, and analyzed the antiviral effects of ICP27 sequence-specific siRNA on HSV-1 replication, in order to lay the foundation for ICP27 to be a new target in the anti-virus therapy.Objectives:1 To established the recombination Vero-siRNAs cell lines expressing the ICP27 sequence-specific siRNAs stably.2 To investigated the antiviral effects of ICP27 sequence-specific siRNA on HSV-1 replication in Vero cells.Methods: 1 Three ICP27 sequence-specific siRNA duplexes were designed and cloned into Lentivirus vector pLKO.1-puro to establish pLKO.1-puro-siRNA.2 The recombination lentivirus was gained by transfecting recombination Lentivirus vectors pLKO.1-puro-siRNA,Δ8.2, VSV-G to 293T cell with Iipofectamine2000.3 After infecting Vero cells by the recombination lentivirus, the positive cells were selected by puromycin. Then the single clones were picked and cultured, and the cell lines expressing ICP27 sequence-specific siRNA were established.4 The recombination Vero-siRNA cells were infected by wild-type HSV-1. Then Realtime PCR was used to detect the inhibitive effect on the expression of HSV-1 ICP27 mRNA, and the protein of ICP27 was detected by Western blot.5 Vero, Vero-siCON, Vero-siRNAl, Vero-siRNA2 and Vero-siRNA3 cell lines were inoculated separately by HSV-1 in 0.1 MOI.24h later, the cytopathic effect (CPE) of each cell group were observed. At the same time, collected the cells and supernatant. The dose of virus was determined by TCID.6 The data was analysised by one-way analysis of variance (ANOVA).Results:1 ICP27 sequence-specific siRNAs were successfully cloned into pLKO.1-puro. Restriction enzyme digestion and sequencing showed recombinant lentiviral vector plasmid pLKO.1-puro-siRNAs were correct.2 The cell lines expressing ICP27 sequence-specific siRNA were selected by puromycin selecting and limiting dilution.3 After inoculation of HSV-1, ICP27 mRNA decreased by 85% in the recombination Vero-siRNA cells (P<0.001). Also the expression of the ICP27 protein was less than the control groups.4 The number of the cytopathic effect (CPE) in siRNAs cells was 40 percent less than that in vero and vero-siCON cells. TCID50 results admired that the dose of virus from siRNAs cells was lower 1-2 log10 than that from Vero and Vero-siCON cells (P<0.001). It was also displayed that ICP27 sequence-specific siRNAs could inhibit the replication of HSV-1 in vitro, and the inhibition ratio was more than 80 percent, which of siRNA2 was the highest, 97%.Conclusions:1 The recombination cell lines expressing the ICP27 sequence-specific siRNA was successfully constructed.2 SiRNA could effectively inhibit the replication of HSV-1 in vitro through silencing ICP27.