The Effect Of RNA Interference Targeting ICP4 Expression On HSV-1 Replication

Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus with envelope, it infects widely among people. HSV is typically neurotropic, which may enter the central nervous system by reverse axonal transfer after infecting peripheral neurons and establish latent infection, the latter always leads to severe injuries in the central nervous system by lysis. The HSV genome is consist of immediate early genes (IE), early genes (E) and late genes (L), which are expressed strictly in sequence. The immediate early protein 4 (ICP4), one of IE gene products, can stimulate the viral early gene expression and DNA synthesis, and induces the expression of viral late gene, thus ICP4 has been regarded as one key activator of HSV-1 gene expression and infection. RNA interference (RNAi) is a phenomenon of self-protection of cell against the violations of the foreign genes, it has been employed widely in molecular biological field to knock down specific gene expression during recent years.In this study, firstly, the recombinant eukaryotic lentivirus plasmid pLKO-puro-ICP4-siRNA which had siRNA targeting ICP4 was constructed. Then the recombinant Vero cell lines which stably expressed ICP4 siRNAs were established by infecting Vero cells with the recombinant lentivirus, in which ICP4 expression was effectively knocked down. Further, the inhibition effect of HSV-1 replication was assayed by HSV infecting siRNA expressed Vero cells. This study tried to provide possibility of the treatment of HSV-1 diseases by RNAi in the future.Objectives:1 To establish the recombinated cell lines expressing the small interfering RNA targeting HSV-1 ICP4.2 To analyze the effect of RNAi targeting HSV-1 ICP4 on the HSV-1 replication.Methods:1 Construct recombinant eukaryotic expression lentivirus plasmid PLKO.l-puro-ICP4-siRNA with genetic engineering, digest and sequence to confirm.2 Construct the recombinant lentivirus PLKO.1-puro-ICP4-siRNA through three plasmids co-transfection of 293T cells.3 Construct the recombinant Vero-ICP4-siRNA cells by the above lentivirus infecting Vero cells and puromycin screening.4 Identify the recombinant Vero-ICP4-siRNA cells with Realtime PCR and Western blot.5 Assay the effect of ICP4 siRNAs on HSV-1 replication by observing CPE of HSV on recombinant vero cells and statistically analyse the titer of HSV-1 with TCID50 after HSV infecting Vero cells.Results:1 Recombinant eukaryotic expression lentiviral plasmid PLKO.1-puro-ICP4-siRNA was successfully constructed.2 ICP4 siRNA expressed lentiviral particles with the effective concentration were produced.3 The Vero cell lines which stably expressing ICP4 siRNA were successfully established.4 ICP4 in the recombinant Vero-ICP4-siRNA cells were inhibited at the mRNA and protein levels.5 The CPE of HSV on Vero-ICP4-siRNA cells was much less than that on control Vero cells under microscopy; and the TCID50 of HSV infecting Vero-ICP4-siRNA cells was remarkably reduced compared to control Vero cells by ANOVA(P<0.001); further more, the inhition effect of siRNA targeting two ICP4 sites were stronger than that of siRNA targeting one single ICP4 site (both P<0.001).Conclusions:1 The recombinated cell lines expressing the siRNAs targeting ICP4 were successfully constructed.2 The siRNA targeting ICP4 significantly inhibites the expression of HSV-1 replication, and the siRNAs targeting two sites have synergistic inhibitory effects.