Viruses and RNA interference in mammalian cells

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supported the idea that RNAi is a mechanism, which protects mosquitoes from viral infection (Keene et al., 2004). RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. It was also shown that the central catalytic component of the RISC, the nuclease Ago-2, is necessary for antiviral defense in adult Drosophila melanogaster. Increased mortality in ago-2–defective flies, which were infected with Drosophila C virus (DCV), and with Cricket Paralysis virus (CrPV) was associated with a dramatic increase in viral RNA accumulation and virus titers. The physiological significance of RNAi as antiviral mechanism is emphasized by finding that DCV encodes the RNAi potent suppressor. This suppressor binds long dsRNA and inhibits processing of dsRNA into siRNA, but does not bind short siRNAs or disrupt the miRNA pathway. Based on these results it was suggested that RNAi is a major antiviral immune defense mechanism in Drosophila. Whether RNAi has a similar function in mammals is under intense investigation (van Rij and Andino, 2006). Invertebrates Along with insect studies, successful researches of Caenorhabditis elegans have been made. C. elegans contains a single Dicer gene. Similar to plants, C. elegans also supports transient RNAi and systemic RNAi (Voinnet, 2005). It was shown that multiple genes of C. elegans required for RNAi are also required for resistance against vesicular stomatitis virus (VSV). VSV was engineered to encode a EGFP (enhanced green fluorescent protein) fusion protein; two types of cells were infected: wild type and cells lacking components of the RNAi. The RNAi-defective cells produced more EGFP and infectious particles, establishing that RNAi of invertebrates is a part of an antiviral defense (Wilkins et al., 2005; Schott, et al., 2005). 16
4. RNA interference in mammals The problem, whether there is mammalian antiviral RNAi counterpart, is the subject of many studies. It was supposed, that RNA silencing machinery is not typical for mammalian cells. This skepticism was derived from the fact that mammalian cells respond to the presence of dsRNA with activation of IFN system. Plants and invertebrates do not possess this system; hence it might functionally replace the RNA interference (Gitlin and Andino, 2003). Then scientists discovered that siRNAs themselves are not able to induce IFN system. (Elbashir et al., 2001). This finding entailed other question whether siRNAs could target mammalian virus and whether viruses would have means to avoid such an onset (Schütz and Sarnow, 2006). It was considered, that mammalian cells might not need to interfere with the RNAi pathway, because they have an effective IFN, which responds to the accumulation of viral dsRNAs by inducing the synthesis of a large group of genes which exert inhibitory effects on viral gene expression (Gale et al., 2000; Katze et al., 2002). Viruses have evolved highly sophisticated mechanisms for interacting with the host cell machinery, and recent evidence indicates that these interactions also involve RNAi pathways. Last years the evidences that RNAi functions as an antiviral defense mechanism in mammalian cells are accumulating. To the present time, there are only few papers describing the mechanism of antiviral action of RNAi in mammalian cells. Artificially triggered RNAi The mammalian cellular RNAi machinery can inhibit virus replication, when the formation of siRNAs is experimentally induced (Li et al., 2004). To attest this supposition, the experiment with poliovirus replication, sense ssRNA virus, in the presence of siRNAs was performed. The poliovirus’ genome is identical to mRNA, therefore can immediately initiate translation of virus-encoded proteins. In one-step growth curves artificial siRNAs resulted in a 100-fold reduction of the progeny titer (Gitlin et al., 2002). But the first RNAi-mediated suppression of mammalian virus replication in tissue culture has been shown on Coxsackievirus B3 (CVB3), which is the most common causal agent of viral myocarditis. CVB3 belongs to the Picornaviridae family and has a ss(+)RNA genome (Yuan et al., 2004). Also the down regulating viral gene expression and replication of negative- and positivestrand RNA viruses were demonstrated by the examples of influenza A and West Nile virus 17
Page 1 and 2: UNIVERSITY OF TARTU FACULTY OF SCIE
Page 3 and 4: ACRONYMS AND ABBREVIATIONS CrPV - C
Page 5 and 6: LITERATURE OVERVIEW 1. Intracellula
Page 7 and 8: RNAi The development of biotechnolo
Page 9 and 10: Figure 3. RNAi mechanism. RNAi is t
Page 11 and 12: Figure 4. Dicer components. 2 helic
Page 13 and 14: Link between miRNA, siRNA and RNAi
Page 15: 3. RNAi as an antiviral defense in
Page 19 and 20: diseases, including liver cirrhosis
Page 21 and 22: esponse by suppressing the activity
Page 23 and 24: CONCLUSIONS In the present bachelor
Page 25 and 26: REFERENCES Aagaard, L. and Rossi, J
Page 27 and 28: Gitlin, L., Karelsky, S. and Andino
Page 29 and 30: Li, W.X. and Ding, S.W., (2001) Vir
Page 31 and 32: Schütz, S. and Sarnow, P. (2006) I

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