Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication.
The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes.
Sriram
Bandi
B
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Thakral
Deepshi
D
Panda
Subrat Kumar
SK
D001483:Base Sequence; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D016752:Hepatitis E virus / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
D016337:RNA, Catalytic; D012367:RNA, Viral; C427204:hammerhead ribozyme