Construction of Ribozyme Aimed at Hantaan Virus S Genome Segment and Demonstration of Function as Anti-viral Drug

ABSTRACT

Hantaan virus is the causative agent of rather severe form of hemorrhagic fever with renal syndrome which occurs widely in north-eastern Asia including Korea, China and far eastcrn part of Russia. Although several types of vaccine for this disease have been developed, the therapeutic agent has not been developed yet. Therefore, we launched the construction of ribozyme to be used as the therapeutic purpose of the disease. Ribozyme which cleaves RNA as an enzyme is a RNA oligonucleotide specific to target RNA. We constructed a ribozyme oligonucleotide aimed at S genomic RNA segment of Hantaan virus (strain 76-118) containing T7 promoter region cornplementary to promoter primer oligonucleotide. Then two oligonucleotides were annealed to prepare double stranded transcription template, and transcription was performed in vitro. Thus, we could prepare the clone of whole S segment of the virus by RT-PCR, and then BamHI/HinCII fragment of the S genome segment was subcloned to pT7T319U vector containing T7 promoter in genome sense. The substrate transcript was made by run-off transcription. These substrate and ribozyme transcripts were used to detect cleavage activity of the ribozyme to the target RNA substrate prior to its application to cultured cell. The cleavage reaction showed that the ribozyme cleaves the target RNA which is S segment of Hantaan virus. To know whether the ribozyme works in cell infected with Hantaan virus as well, the ribozyme was transfected to Vero-E6 cell by lipofectin after inoculation of the virus. The transfected ribozyme was detectable in the cell by RT-PCR utilizing ribozyme specific primers. On 7 days after inoculation, the culture media were harvested and used to determinate viral titers by immunoenzyme plaque assay. In contrast to the mock transfected negative control, the viral titers of the cultures transfected at 1, 2 and 3 days after the virus inoculation were lowered to 1/100 level. This result suggests that the ribozyme inhibits the multiplication of Hantaan virus in cultured cell successfully in early stage of infection, and ribozyme is a possible new anti-viral drug against the virus infection.