Research Article: Engineered External Guide Sequences Are Highly Effective in Inhibiting Gene Expression and Replication of Hepatitis B Virus in Cultured Cells

Date Published: June 12, 2013

Publisher: Public Library of Science

Author(s): Zhigang Zhang, Gia-Phong Vu, Hao Gong, Chuan Xia, Yuan-Chuan Chen, Fenyong Liu, Jianguo Wu, Sangwei Lu, Haitao Guo.


External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy.

Partial Text

Nucleic acid-based gene interfering technologies, such as antisense oligonucleotides and RNA interference (RNAi), have been shown to be a promising gene targeting approach for use in basic research and clinical therapeutic applications [1]. Ribonuclease P (RNase P) has been found in all organisms examined and its enzymatic activity is responsible for the maturation of 5’ termini of all tRNAs which account for about 2% of total cellular RNA [2–4]. This enzyme is a ribonucleoprotein complex and catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA (Figure 1A) [2–4]. Studies on RNase P substrate recognition revealed that the enzyme recognizes the structure rather than the primary nucleotide sequence of the substrates, and can cleave a model substrate that contains a structure equivalent to the acceptor stem, the T-stem, the 3′ CCA sequence, and the 5′ leader sequence of a ptRNA molecule (Figure 1A) [5]. Altman and colleagues proposed that RNase P can be recruited to cleave any mRNA using a custom-designed external guide sequence (EGS) that hybridizes with the target mRNA to form a structure resembling a tRNA substrate (Figure 1B–D) [6,7]. EGS RNAs derived from natural tRNA sequences can be effective in blocking gene expression in bacteria and in mammalian cells [7–10]. EGSs have been shown to inhibit HIV gene expression and replication in human cultured cells [11]. Furthermore, we have shown that EGSs that were derived from a natural tRNA effectively induced human RNase P to cleave the mRNAs of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) in vitro [10,12,13]. A reduction of ~75% in HSV and HCMV gene expression was observed in viral infected cells that expressed these functional EGS RNAs.

The EGS-based technology represents an attractive approach for gene inactivation since it utilizes endogenous RNase P to generate highly efficient and specific cleavage of the target RNA [3,40]. However, little is known about the rate-limiting step of the EGS-targeting approach in cultured cells. Equally unclear is whether the efficacy of the EGSs can be improved, and if so, how it can be improved. In this study, we constructed EGSs that target an accessible region of HBV S mRNA. Our results indicated that an EGS variant, S-C386, is about 50 times more active [Vmax(apparent) /Km(apparent)] in directing RNase P to cleave the S mRNA sequence in vitro than S-SER, an EGS derived from the natural tRNASer sequence. Moreover, S-C386 inhibited HBV gene expression (i.e. HBV transcripts and HBsAg/HBeAg) in cultured cells by about 97-98% and was more effective than S-SER, which reduced HBV gene expression by about 75%. In contrast, a reduction of less than 10% in HBV gene expression and DNA replication was observed in cells that expressed control EGS S-C386-C, S-SER-C, or TK112. S-C386-C and S-SER-C exhibited similar binding affinity to s38 as S-C386 and S-SER, respectively, but were inactive in directing RNase P-mediated cleavage due to the presence of the mutations at the T-loop that precluded RNase P recognition (Figures 1 and 2Table 1). Our results suggest that the observed reduction in viral gene expression and DNA replication with S-C386 and S-SER is primarily attributed to the specific targeted RNase P-mediated cleavage induced by these two EGSs as opposed to the antisense effect or other nonspecific effects of the EGSs. Moreover, our results suggest that the EGS (i.e. S-C386) that is more active [Vmax(apparent) /Km(apparent)] in inducing RNase P to cleave the S mRNA sequence in vitro is also more effective in inhibiting HBV gene expression and DNA replication in cultured cells and that increasing the activity of EGS in directing RNase P cleavage in vitro may lead to improved efficacy in inhibiting gene expression in cultured cells.