1. Viral Silencing Suppressors. Tools forged to fine-tune host-pathogen coexistence

2. Presentation by Seema Hassan Satti Ph.D 1 st semester 00-arid-1057 Botany Department

3. INTRODUCTION RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing.

4. Plant Viruses Plant viruses are amongst the most important pathogen causing huge economic losses by reducing crop quality and quantity. A better understanding of the viral infection processes and plant defense strategies is important for crop improvement.

5. Classification of Viruses Based on their genome organization, viruses can be classified as; Positive-sense-, Negative-sense-, Double-stranded-RNA viruses Single-stranded or double-stranded-DNA viruses

6. Mechanism of Infection Viral RNA or DNA encode for surprisingly restricted number of proteins that coordinate the infection process, Viral proteins interact with host factors to manipulate biochemical events and molecular interactions required for the virus replication. Viruses can spread within the plant through plasmodesmata or through phloem.

7. Host defense mechanism A set of complex interactions involving virus attack and host defense develops. These include; Hypersensitive reaction (HR) Systemic acquired resistance (SAR) Activation of ubiquitin/26 s proteosome system (UPS) RNA silencing (RNA interference, RNAi)

8. Gene silencing This term refers to the ability of a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research.

9. RNA Silencing in plants The RNA silencing process in plants consists of following phases; Initiation phase Effector phase Amplification phase

10. Initiation phase During silencing initiation phase double- stranded RNAs (dsRNA) of different origins are processed by an Rnase III type enzyme DICER, (DCR in plants, Dicer-like proteins DCLs) into short 21-24 nt long small RNA (sRNA) duplexes. DICERs require double-stranded RNA binding (DRB) proteins for accurate sRNA production.

12. Effector phase The sRNA s are then stabilized at their 3’ end by Enhancer 1-dependent methylation. These are then exported to be loaded onto Argonaute proteins (the effectors of RNA- induced Silencing Complex RISC or RNA- induced Transcriptional Silencing Complex RITC.

13. ARGONAUTE proteins bind small RNAs and their targets. The Arabidopsis ago1 mutant and the octopus Argonauta argo ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis; ago1 has thin radial leaves and was named for the octopus Argonauta which it resembles.

14. Continued….. Guided by sRNA sequence RISC induces slicing of translational repression of its target RNAs in a sequence specific manner (During PTGS). Whereas, RITC complex causes histones and DNA methylation resulting in transcriptional gene silencing (TGS) of the homologues gene.

15. Amplification Phase In plants the effecter step can result in the amplification of silencing response involving RNA-dependent RNA polymerases proteins.

16. Action of viral suppressors of RNA silencing Some viruses avoid RNA silencing by replication within well defined subcellular compartments like ER spherules. Some replicate and move fast enough to outrun the mobile silencing signals. The most common way to protect viral genome against RNA silencing is to encode proteins that act as suppressors of RNA silencing. These proteins are termed as Viral Suppressors of RNA silencing or VSRs.

19. Blocking of antiviral response This can be achieved through multiple ways like; Dicer proteins or co-factor activity inhibition. dsRNA/siRNA-sequestration. AGO protein destabilization prior of RISC assembly. The most widespread strategy is the ds-si RNA sequestration that is used by VSRs encoded by diverse virus genera (P19, Hc-Pro, P21, p15, p130 etc).

21. P19 Protein Probably the most characterized si RNA binder is the tombusviral P19 protein. Crystallographic studies have shown that P19 head-to-tail homodimer act as molecular caliper to size-select and sequester siRNA duplexes in a sequence-independent manner.

24. Other strategies used by virus Arrest of functional RISC assembly through AGO interaction. Interaction of programmed antiviral RISC complex. VSR activities downstream of RITC and RITS. Modulation of AGO homeostasis. Plant RDR-based activity suppressors.

25. CONCLUSION Due to their varsetality and availability, VSRs are employed in molecular research. The use of P19 provided sensitive detection without the need of amplification. As a tool of unraveling molecular basis of silencing itself. VSR can be employed effitiently to limit transgene silencing and attain high –level expression of diverse products like vaccines, pharmaceuticals, high-nutritive foods and high value products.

26. THANK YOU!!!

27. REFERENCE Csorba, Tibor., Kontra, Levente., Burgyan, Jozsef. Viral silencing suppressors. Tools forged to fine- tune host-pathogen coexistence. 2015. Virology. 479-480. 85-103