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Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein
Author(s) -
Chilakamarthi Ushasri,
Mukherjee Sunil K.,
Deb J.K.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.04.084
Subject(s) - ribozyme , biology , vs ribozyme , hammerhead ribozyme , mammalian cpeb3 ribozyme , control of chromosome duplication , replication factor c , viral replication , dna replication , microbiology and biotechnology , minichromosome maintenance , rna , dna , virology , genetics , gene , virus
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 ∼33% cleavage activity on synthetic rep transcript within 1 h 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 2 h. 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.