The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double stranded (ds)RNA ‘trigger’ molecules into small interfering RNAs (siRNAs) by enzymes of the Dicer family. The siRNA molecules are essential components of the RNA induced silencing complex (RISC), which uses the siRNA sequence to be guided to complementary targets that are subsequently inactivated by the slicing activity of Argonaute proteins, the active component of RISC. To counteract antiviral RNA silencing, plant viruses encode dedicated suppressor proteins. The identified suppressor proteins so far, mostly are encoded by plant positive strand RNA viruses and DNA viruses. This thesis and previous work in our laboratory (Bucher, 2006) centred around the characterisation of the RNA suppressor proteins of negative strand plant RNA viruses. This group of viruses is unique in having a replication cycle in both their botanical host and insect vector, making them likely to encounter antiviral RNA silencing in both types of organisms. At the onset of this thesis research, the suppressor proteins of two negative strand RNA plant viruses, i.e. of Tomato spotted wilt virus (TSWV, genus Tospovirus) and of Rice hoja blanca virus (RHBV, genus Tenuivirus), had been identified, but their mode of action remained unknown. In chapter 2 of this thesis, the RNA silencing suppressor of RHBV, the NS3 protein, was investigated in further detail. Its suppressor action was confirmed in plants and also established in insect cells. Molecular and biochemical analyses of the NS3 protein showed a high affinity for the archetypical 21 nt siRNA molecules, but not for longer dsRNAs. By recruiting these siRNA molecules, NS3 was shown to interfere with the assembly and function of RISC in Drosophila embryo extracts. Sequestration of siRNAs, conserved between the RNA silencing pathways of all eukaryotes, enables RHBV to counteract this antiviral response in its insect vector and plant host. RNA silencing also serves a critical role in gene expression regulation and genome integrity. Key players in this part of the RNA silencing are the microRNA (miRNA) molecules. In addition, the binding affinity of NS3 to unwound miRNA duplexes was proven to be comparable to that of siRNAs, which is in agreement with developmental abnormalities observed in transgenic Arabidopsis plants after constitutive expression of the NS3 protein. Knowing the interference strategy of RHBV NS3, the sequence requirements for siRNA binding were examined in chapter 3. By comparing amino acid sequences of the RHBV NS3 protein to its paralogs of other tenuiviruses, two conserved and predicted surfaced-exposed regions were identified. Deletion of either domain resulted in dysfunctional suppressor proteins while deletion of single alanine substitutions in these regions had no effect on their suppressor activity or siRNA binding capacity. However, when three clustered positively charged amino acids (K173-K175), present in one of these domains, were substituted the siRNA binding affinity of this mutated protein was completely abolished, coinciding with complete lack of suppressor activity. This confirmed the alleged role of siRNA binding as being crucial for the RNA silencing suppression activity of NS3. The suppressor protein (NSs) of tospoviruses was subject of the studies presented in chapter 4. In contrast to tenuiviral NS3, the tospoviral NSs showed size-independent binding to dsRNA. Its ability to bind also longer dsRNA was shown to result in the inhibition of Dicer-mediated processing of longer substrates into siRNAs. In addition, binding of NSs to miRNA duplexes was confirmed in planta. As tospoviruses belong to the large Bunyaviridae family, which also hosts many animal viruses, the observed high affinity for longer dsRNA molecules of their NSs proteins may reflect a common ancestry with such animal viruses. Indeed, for animal infecting viruses the capacity of their host defence antagonistic proteins to bind long dsRNA seems favourable, since these molecules are not only a substrate for Dicer, but are also recognised by alternative innate defence pathways like the interferon response. Although at the time there were few indications for an antiviral activity of the RNA silencing machinery in vertebrate systems, the Influenza virus A NS1 protein scored positive as suppressor of RNA silencing in plant- and insect-based assays (Bucher, 2006; Li et al., 2004). Chapter 5 investigates the potential activity of NS1 as RNA silencing suppressor further, now using homologous (human) cell systems. Thus NS1 is shown not to bind siRNAs but exclusively long dsRNA molecules with high affinity and by doing so it is able to inhibit Dicer activity. Two point mutations in its RNA binding domain, previously implicated in both RNA silencing and the interferon response, resulted in the accumulation of siRNAs in Dicer cleavage assays. Recombinant influenza viruses expressing wildtype (PR8-NS1) or the mutant NS1 protein (PR8-NS1rb) were constructed and the effect on virus replication and accumulation was assayed. This demonstrated that viral titers drastically decreased for PR8-NS1rb compared to PR8-NS1 and since interferon production was not induced during PR8-NS1rb infections, this hinted towards an antiviral role for RNA silencing in mammals. A second line of research underscored this interpretation; wildtype NS1 protein, but not the NS1rb mutant protein, was able to complement a Tat-minus Human immunodeficiency virus-1 (HIV-1) virus. Interestingly, also the NS3 protein of RHBV rescued this HIV mutant, indicating a role of small RNA molecules in vertebrate antiviral silencing. In conclusion it is shown that negative strand RNA viruses of plants encode suppressor proteins that combat RNA silencing by interacting with dsRNA, thereby ensuring interference of this host response in both plant host and insect vector. Having said this, the suppressors of tenuiviruses and tospoviruses do not act in the same way. While tenuiviral NS3 only interferes with RISC assembly, NSs also inhibits Dicer activity. Both strategies enable the suppression of antiviral silencing in their insect vector and plants. Furthermore, the presented data on the NS1 protein of Influenza virus A adds to the recently emerging evidence that also mammalian viruses may encode suppressors to counteract antiviral action of the siRNA or the miRNA pathway.
|Qualification||Doctor of Philosophy|
|Award date||26 Nov 2007|
|Place of Publication||[S.l.]|
|Publication status||Published - 2007|
- gene expression
- rna viruses