Abstract
An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.
Highlights
Herpes simplex virus 1 (HSV-1), a member of the human herpesvirus family, is one of the leading causes of viral infections in humans and reactivation of the virus from latency can cause herpes simplex encephalitis and corneal blindness in immunocompromised individuals[1]
A position, 66 nts downstream from the start codon was chosen as the cleavage site for the external guide sequence (EGS) since it was among the regions where the ICP4 mRNA was most modified by dimethyl sulphate (DMS)
EGS-mediated degradation of mRNA represents a new nucleic acid based approach for gene silencing applications. This strategy is a promising approach because intracellular ribonuclease P (RNase P) can be guided with custom-designed EGSs to cleave any target mRNAs efficiently and specifically[3,24]
Summary
Herpes simplex virus 1 (HSV-1), a member of the human herpesvirus family, is one of the leading causes of viral infections in humans and reactivation of the virus from latency can cause herpes simplex encephalitis and corneal blindness in immunocompromised individuals[1]. EGSs have the potential to be an effective therapeutic approach for human diseases such as HSV-1 infection[16,17,18] Increasing both the in vitro and in vivo activity of EGSs can contribute to the efficiency of the reaction of EGS-induced cleavage of target RNA by RNase P. We determined the activity of the constructed EGSs in guiding RNase P to cleave the target HSV-1 ICP4 mRNA sequence and their efficacy in inhibiting HSV-1 gene expression and viral replication in HSV infected cells. C468-A, an EGS variant, showed 60 times more efficient in guiding RNase P to cleave ICP4 mRNA sequence than SER-A derived from a natural pre-tRNA sequence. Our results demonstrate that EGSs are effective in inhibiting HSV-1 growth in cultured cells
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