Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein.
Geminiviruses pose serious threat to many economically important crops such as mungbean, tomato, cotton, etc. To devise a specific antiviral strategy at the viral DNA replication level, a hammerhead ribozyme was directed against the mRNA of the replication initiator protein (Rep). Rep is the most important viral protein for the DNA replication of the Mungbean yellow mosaic India virus (MYMIV), a member of the Geminiviridae family. The ribozyme showed approximately 33% cleavage activity on synthetic rep transcript within 1h under in vitro conditions, whereas the mutant ribozyme, designed to lack the catalytic activity but target the same site, showed no cleavage. The in vivo efficiency of ribozyme was evaluated in Saccharomyces cerevisiae as it can act as a surrogate host for replication of the MYMIV-DNA and lacks RNAi machinery. In the presence of the ribozyme, growth of the yeast cells that are dependent on geminiviral replication was inhibited by 30% and cellular generation time was increased by 2h. The RT-PCR analysis showed a maximum of about 50% reduction in the rep mRNA level in presence of the ribozyme compared to its noncatalytic mutant control. About 65% decrease in geminiviral DNA replication was observed due to the downregulation of replication initiator protein by the ribozyme. These results raise the possibility of engineering resistance to geminiviruses employing the ribozyme approach.
Chilakamarthi
Ushasri
U
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, India.
Mukherjee
Sunil K
SK
Deb
J K
JK
D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017791:Geminiviridae / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000821:virology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases