Viruses and RNA interference in mammalian cells

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RNA silencing suppressors The fact that B2 of both the Nodamura virus (NoV) and the Flock House virus (FHV) can inhibit RNAi triggered artificially in human cells was proved by scientists in 2005 (Sullivan and Ganem, 2005). The NoV is not enveloped ss(+)RNA virus. It occurs in nature as an unapparent infection of mosquitoes in Japan, also asymptomatically infects pigs (Bailey et al., 1974). Many viral RSS identification in mammalian cells include the use of shRNAs. Scientist developed a highly sensitive assay of RNAi in mammalian cells, which shows that RNAi triggered by either shRNAs or siRNAs, can be inhibited by the NoV B2 protein. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RISC-processed RNAs, inhibiting the Dicer cleavage reaction. Both endogenous genes and transgenes encoding EGFP or luciferase have been used as the reporter target gene in RNAi suppression assays. The assay based on RNAi of a destabilized EGFP (dEGFP) reporter gene was developed to identify inhibitors of RNAi in mammalian cells (Sullivan and Ganem, 2005). Less EGFP was detected, when cotransfected with the anti-EGFP shRNA than, when co-transfected with the control anti-luciferase (anti-Luc) irrelevant shRNA. The facts, that transfected siRNAs could suppress viral replication by reducing viral RNA accumulation and that viral RSSs function in mammalian cells, could enhance the evidence, that mammalian RNAi machinery possesses an antiviral response (Gitlin et al, 2002). VP35 protein of Ebola virus has RSS activity, which is functionally equivalent to that of the HIV-1 Tat protein. VP35 can replace HIV-1 Tat and thereby support the replication of a Tatminus HIV-1 variant. It shows that viruses need to counteract RNAi in order to replicate. Also VP35 protein is known as IFN antagonist. The VP35 dsRNA-binding domain is required for this RSS activity. (Haasnoot et al., 2007) Influenza A, B, C viruses NS1 protein and vaccinia virus E3L protein were demonstrated to be essential proteins that suppress RNA silencing in Drosophila. The major fact is, that these mammalian virus proteins, are also distinct dsRNA-binding proteins essential for pathogenesis by inhibiting IFN antiviral response. It was also demonstrated that the dsRNAbinding domain of NS1 is essential and sufficient for RNAi suppression (Li et al., 2004). Influenza A virus NS1 protein and vaccinia virus E3L protein are also capable to replace the HIV-1 Tat RSS function (Haasnoot et al., 2007). The adenovirus virus-associated RNAs I and II (VA-RNAI and II) are also capable of inhibiting RNAi antivaral response. VA RNA are highly structured RNAs, about 160 nt in length, encoded by adenoviral genome and expressed by host RNA polymerase III. (Mathews and Shenk, 1991). At late infection human adenovirus inhibits RNAi antiviral 20
esponse by suppressing the activity of Dicer and RISC. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. The siRNAs, received from VA RNAI and VA RNAII are incorporated into active RISC. A panel of mutant adenoviruses defective in virus-associated (VA) RNA expression was used to detect the mechanism by which adenovirus blocks RNAi. The investigation results suggested that the adenovirus VA RNAs antagonize the cellular defense pathways directed against both long (interferon-induced) and short (RNAi-induced) dsRNA by binding and inactivating PKR and Dicer enzymes VA RNAI potently inhibited RNAi induced by endogenously transcribed pre-miRNAs and shRNAs and weakly inhibited RNAi induced by in vitro transcribed, transfected shRNAs. But it did not affect RNAi induced by artificial siRNA-miRNA duplexes. Also it was observed, that VA RNAI expression inhibited the production of mature miRNAs from premiRNA precursors, because VA RNAI is a potent inhibitor of pre-miRNA or shRNA nuclear export, competing for a limited pool of the cellular Exp5 nuclear export factor. To conclude, adenovirus VA RNAI is a gene product, which is able to specifically block RNAi in human cells (Lu and Cullen, 2004) These findings support the hypothesis that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication (Haasnoot et al., 2007). Virus-encoded miRNAs The fact, that viruses use the RNAi machinery serves as another evidence of its existence. Viruses encode miRNA-like molecules, that can potentially affect cellular miRNA processing and functioning (Berkhout and Haasnoot, 2006) Virus-encoded miRNAs were identified while cloning the small RNA profile in cells infected with Epstein–Barr virus (EBV). This is a large DNA virus, which belongs to the herpesvirus family (Pfeffer et al., 2004). Other members of the herpesvirus family and other viruses with large DNA genomes could encode miRNAs to exploit RNAi for the regulation of host and viral expression. (Berkhout and Haasnoot, 2006). Processing of virus-encoded miRNAs is going through the same pathway as host miRNAs. The early miRNAs are cleaved by the late miRNA at the predicted position in the replication cycle, thus reducing early gene expression, and possibly evading immune recognition of the infected cell. The viral miRNA targets the early transcript for degradation. Viruses exploits the RNAi machinery to regulate their gene 21
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 and 16: 3. RNAi as an antiviral defense in
Page 17 and 18: 4. RNA interference in mammals The
Page 19: diseases, including liver cirrhosis
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

Viruses and RNA interference in mammalian cells

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