Objective: The vaccines currently developed against infectious diseases fail to induce effective antiviral immune responses to control an abrupt outbreak of viral diseases epidemic in a short space of time. Hence there is an urgent need to develop emergency vaccines capable of producing prophylactic effects against infectious diseases. RNA interference (RNAi) is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes. The rapidity and uniqueness of RNAi responses can make up for the current inadequacy of antiviral preventive regimes. Here we evaluate the antiviral potential of short hairpin RNA (shRNA) targeting C (core) gene of hepatitis B virus (HBV). It plays essential roles in HBcAg encoding and viral attachment to susceptible cells during its life cycle. The present study was intended to investigate the inhibitory effect of C-specific shRNAs on HBV replication and expression in BHK-21 cells.
Methods: The reporter gene expression vector of pC-EGFP-N1 was constructed by cloning the DNA (PCR product) of HBV C into the EcoRI-HindIII sites of pEGFP-N1 to fuse C to enhanced green fluorescent protein (EGFP) for providing a reporting system for monitoring siRNA function. Plasmid pC was constructed by cloning the DNA of HBV C into the EcoRI-HindIII sites of pCDNA3.1B(-) directly under the control of cytomegalovirus promoter. Two plasmids (S1 & S2) were constructed to express shRNAs targeting C of HBV with the length of 24 nucleotide (nt) homologous in sequence to the HBV C gene. Plasmids were designed and synthesized according to the HBV genome (HBV genotype B, ayw1 subtype) of chronic hepatic B patients from 56 ethnic minorities in China. After cloning and sequencing, the investigators registered them with the GenBank accession numbers of AY517488 (CYN/2002) and AY517489 (CYN/2000), etc. Simultaneously, one of nonspecific shRNA-S3 with a length of 24 nt was also designed randomly for negative control. After cloning into vector pU6 for constructing shRNA-expressing plasmids, they were then cotransfected into BHK-21 cells along with reporter gene expression vector of pC-EGFP-N1. First, upon a determination of the number of cells exhibiting EGFP expression in BHK-21 cells as detected by an Olympus BH-2 fluorescence microscope and FACS-440 flow cytometry (Becton-Dickinson, USA) at different times after cotransfection, the investigators evaluated the gene inhibitory efficiency of both plasmids by an EGFP reporter system in BHK-21 cells. Subsequently, the antiviral efficacy of both plasmids in BHK-21 cells was further investigated by real-time quantitative polymerase chain reaction.
Results: In comparison with single plasmid transfection pC-EGFP-N1 or pEGFP-N1, fluorescence microscope and flow cytometry detection at 24 h post-cotransfection revealed that the expression of reporter gene EGFP in cotransfection group involving S1 or S2 and S1 + S2 cotransfection group was reduced significantly by about 90% in EGFP signal versus the control. And the EGFP expression in control plasmid cotransfected S3 or pU6 was not significantly reduced in BHK-21 cells (P < 0.01). It was found that the expression of mRNAs of HBV C and EGFP gene as detected by real-time quantitative PCR was the same as that detected by fluorescence microscope and flow cytometry (P < 0.01), thereby further corroborating the antiviral efficacy of RNAi. The antiviral efficacy extended to almost 48 hours. The results indicted that a DNA vector-based RNAi technology could effectively and specifically inhibit the replication and expression of HBV in BHK-21 cells.
Conclusion: For the first time it has been found that RNAi induced by shRNA targeting C gene exerts an effective and unique inhibition of HBV replication and expression in BHK-21 cells. Thus RNAi may provide an effective emergency vaccine against major infectious diseases such as HBV infection.