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Role of ribozymes in viral infection
1. Role of ribozymes in viral infection
Presented by:
Somvir Singh
17 BCM-04
2. Contents
• Ribozymes
• Catalytic activity of ribozymes
• History
• Role in viral infection
• Type of ribozymes
• Design of ribozymes
• Delivery Systems
• Monitoring
• Targets
• Conclusion
3. • Ribozymes (ribonucleic acid enzyme) are the
RNA molecules that are capable of performing
specific biochemical reactions.
Ribozymes
4. • Ribozymes cleave mRNA molecules in a sequence specific,
catalytic manner.
Catalytic activity of ribozymes
5. History
• The term ‘ribozyme’ was first introduced by Kelly kruger et al.
in 1982.
• All known enzymes were considered as protein until the
discovery of ribozymes.
• Ribozymes were discovered by Thomas Cech and Sidney
Altman in 1980s.
• Thomas Cech- worked on Tetrahymena thermophila.
• Sidney Altman-worked on RNase P in E.coli.
6. Role in Viral Infection
• Ribozymes act as promising anti-viral agents.
• They reduce the viral burden through specific targeting and
cleavage of viral genome.
• Mostly hammerhead, hairpin and Rnase P ribozymes are used.
7. Hammerhead ribozyme (HHRZs)
• 30 nucleotide long autocatalytic RNAs, cleaves single-stranded
RNA.
• Found in virus-like elements called virusoids.
• Used to cleave many RNA targets in vitro.
• Used to suppress the effect of Chikungunya virus.
8.
9. • These ribozymes have been developed mainly for antiviral
applications.
• A hairpin ribozyme targeting G575 of the Sindbis virus
genomic RNA was designed.
• Transfected cells, expressed low levels of a constitutive
transcript containing the ribozyme plus recognition
sequences for Sindbis RNA replicase.
• Upon infection with ribozyme, transcript was amplified to
high levels by the viral replicase, resulting in decreased viral
production from infected ribozyme-expressing cells.
Zhang and Burke, 2005.
Hairpin ribozyme
10. Ribonuclease P (RNase P)
• RNase P acts an endonuclease enzyme, involved in generating
the mature 5’ end of t-RNA using pre-tRNA as substrate.
• M1GS ribozyme was used to target overlapping regions of IE
1 and IE 2 mRNA of Human Cytomegalovirus (HCMV) and
thymidine kinase mRNA of Herpes simplex virus-1 (HSV-1)
• Inhibits the infection caused by HCMV and HSV-1.
13. Selection of accessible sites
• RNA adopts a complex secondary structure.
• Traditionally, site selection is based on computational
secondary-structure prediction of the target RNA.
• In-vivo mapping with dimethyl sulphate (DMS) or nuclease
footprinting is extensively used to determine the accessibility
of mRNA to a ribozyme.
14. Selection of variants of ribozymes
• Introduction of mutations.
• E.g., a point mutation in RNase P catalytic RNA from E.coli
(G95->U95) increases the rate of cleavage, whereas another
mutation at nucleotide 200 (A200->C200) enhances substrate
binding efficiency of the ribozyme.
• In-vitro selection of most stable and efficient variant ribozyme.
15. Expression Cassette
• Most important element is the promoter which regulates
expression of the gene construct.
• Ribozyme can be linked to tRNAlys, which is the primer used
in reverse transcription of HIV-1 genome.
17. Monitoring
Efficiency of the ribozyme action can be monitored by:
• Detecting the presence of target mRNA using Northern
Blotting.
• Detecting the presence of its protein products via ELISA,
Western Blotting etc.
• Detecting the presence of target virus by counting the plaque
forming units (PFU).
18. Targets
• Clinical trials are been conducted against human immunodeficiency
virus-1 (HIV-1), HSV-1, hepatitis C virus (HCV) , HBV etc.
• HIV-1-conserved regions like the LTRs, tat, rev mRNA.
• Hepatitis C Virus- M1GS of RNase P is used to target the 5’-UTR of
HCV.
• Herpes Simplex Virus 1-RNase P catalytic RNA directed against
mRNA of major transcription activator ICP4 and thymidine kinase.
19. Conclusion
• Ribozymes are capable of specifically cleaving RNA
molecules, a property which enables them to act as potential
antiviral agents.
• However considerable work is still needed to be done to
enhance our understanding of the mechanism of ribozyme
cleavage in cell with improved delivery methods and to
optimize conditions for application in anti-viral therapy.
In the absence of Rev, mRNAs of the HIV-1 late (structural) genes are retained in the nucleus, preventing their translation.
tat is a protein that is encoded for by the tat gene in HIV-1.[1][2] Tat is a regulatory protein that drastically enhances the efficiency of viral transcription.
The LTRs are partially transcribed into an RNA intermediate, followed by reverse transcription into complementary DNA (cDNA) and ultimately dsDNA (double-stranded DNA) with full LTRs. The LTRs then mediate integration of the retroviral DNA via an LTR specific integrase into another region of the host chromosome.
the three prime untranslated region (3'-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon. An mRNA molecule is transcribed from the DNA sequence and is later translated into protein. Several regions of the mRNA molecule are not translated into protein including the 5' cap, 5' untranslated region, 3' untranslated region, and the poly(A) tail. The 3'-UTR often contains regulatory regions that post-transcriptionally influence gene expression.
