The Role of RNAi in Mammalian Cells in Response to Sindbis virus infection

Donaszi-Ivanov, Andras (2013) The Role of RNAi in Mammalian Cells in Response to Sindbis virus infection. Doctoral thesis, University of East Anglia.

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Viruses are obligate intracellular parasites that need to interact with their host in order to replicate successfully. The understanding of this complex interaction between host and virus is essential for developing new therapeutic strategies as viral infections pose a serious challenge in healthcare, and also because viruses impose enormous costs on the economy. This study focused on the role of RNA interference in the interaction between an RNA virus (Sindbis virus) and its mammalian host cell.
A sensitive image–based viral replication assay was developed to follow Sindbis virus replication in HEK293C cells and the main anti-viral innate responses to virus infection described. Virus replication increased in Dicer and RNA helicase A (RHA) knockout cells, but not when the central regulator of interferon synthesis IRF3 was knocked out. High-throughput Solexa Illumina sequencing was used to detect small viral RNA (svRNA) in Sindbis virus (SINV) infected human cells, and to detect changes in the cellular microRNA (miRNA) expression profile. Very few vsRNA sequences were detected by sequencing during virus infection, and I argue that they are random degradation products, not Dicer-generated svRNAs. Due to the very low level of svRNAs, these were undetectable using northern blotting. We have also found that the expression profile for cellular miRNAs did not change in the early stages of virus infection according to the sequencing data, a finding which was verified by northern blotting. A functional RNAi assay was developed to assess the activity and function of the RNAi system in cells subjected to cellular stress, type I interferon, infection and dsRNA, and northern blotting was used to verify the sequencing data. I have found that certain stress signals -double stranded RNA and SINV infection- decrease the efficiency of siRNA knockdowns in a siRNA-based knockdown assay system.
I have identified two host factors important in Sindbis replication (Dicer, RHA). The lack of vsRNA fragments led to the conclusion that during virus infection the siRNA pathway is suppressed by either the cell or the virus itself, although SINV has been shown not to have any RNAi suppressors in previous studies conducted on its insect vector. This can be explained by the fact that both RNAi and the innate immunity detect the same molecule, dsRNA, placing these two systems into direct competition for the same substrate. My hypothesis is that the siRNA pathway of RNAi is suppressed so that Dicer does not process the long dsRNA into small, 21nt fragments, which are invisible to the innate immune system.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Users 2259 not found.
Date Deposited: 06 Mar 2014 14:02
Last Modified: 31 May 2014 00:38


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