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Cap-snatching is a unique process to facilitate viral genome transcription initiation for segmented, negative stranded RNA viruses (NSVs). For the nuclear-replicating Influenza viruses, cap-snatching occurs at RNA polymerase II (polII) sites, where the viral transcriptase complex, bound at the C-terminal domain of the host RNA polII, is able to directly access to 5’ capped leader sequences from nascent mRNAs. At the start of this thesis research, a few studies pointed to cytoplasmic P bodies as a source from where the cytoplasmic NSVs would steal capped-RNA leaders. However, there were still questions that could not be answered and pointed to a situation that was likely more complex (Chapter 1). In order to better understand the various cytoplasmic RNA granules and indicate their potential as a source of cap donors for the cytoplasmic-replicating NSVs, an overview was made of the different cellular RNA granules and their functions in the regulation of cellular processes and control of cell development (Chapter 2). In addition, the interplay of these granular structures with viruses during an infection, as well as their pro- and antiviral activity was described.
To get a first glimpse of the source of host-derived capped-RNA leaders, an RNA-seq experiment was performed to analyse the host-derived capped-RNA leaders at the 5’ end of viral mRNAs, and identify their corresponding host genes (Chapter 3). Whereas viral transcripts were identified within the transcriptome, capped RNA leader sequences derived from host cellular mRNAs were hardly collected. Likely, their absence resulted from the experimental approach to make a cDNA library, and failure to copy complete 5’ UTR sequences up to the 5’cap. On the other hand, the transcriptome data revealed a change in the gene expression profile induced upon viral infection, in which genes corresponding to the photosynthesis pathway were downregulated the most (Chapter 3).
Earlier studies revealed a colocalization of the hantavirus Sin nombre virus (SNV) N protein with P bodies (PBs), and an affinity of this protein to 5’ caps. Therefore, it was postulated that this affinity likely led to a localization at foci enriched for capped RNA besides P body. To test this hypothesis for bunyaviruses and analyse for generic features, in situ localization studies were performed on bunyaviral N proteins from distinct animal- and plant infecting viruses. The results showed that the N proteins analysed all co-localized with PBs and SGs (Chapter 4). Furthermore, for Tomato spotted wilt virus (TSWV) and Schmallenberg virus (SBV) this was confirmed to occur during a natural infection as well. Interestingly, a preference of TSWV and SBV N protein was observed for the PB-SG docking complex. When the PB enzymatic factor DCP5 gene was silenced, slowing down the de-capping and degradation of mRNA, viral titres went up. However, when the NMD pathway factor UPF1 gene was silenced, assumed to reduce the influx of non-functional mRNAs into PB, viral titres went down only slightly. When the SG formation-related factors G3BP1-like and Rbp47 were silenced, viral titres increased. When the translation initiation factors eIF4E and eIF4A (SG resident components) were silenced, viral titres were positively or negatively affected, respectively. Upon simultaneous silencing of both DCP5 and G3BP1-like genes, TSWV was able to replicate better than in control plants, but less to a lesser extent than when the additive effect of the individually silenced genes. Altogether, these results indicated that the role of PBs as the only/major source from where these viruses could collect capped RNA could be questioned. To test for the use of possible upstream sources of cytosolic capped RNAs, the localization of the TSWV N protein was analysed relative to the perinuclear region. A co-localization of N with RanGAP2, a nuclear envelope and nucleocytoplasmic shuttling factor, was observed, while the N protein also seemed to interact with a particular domain of RanGAP2. When both RanGAP homologs in plants were silenced, TSWV titres went clearly down, pointing to the nuclear pore complex as a potential site from where these viruses could access capped-RNA leaders from the nuclear mRNA efflux (Chapter 4).
As a complementary approach and alternative to find clues that would point to the source of host-derived capped-RNA leaders, high throughput sequencing (HTS) was applied on a large number of capped-leader sequences from viral mRNAs (Chapter 5). By bioinformatic analysis, capped-leader sequences were analysed and their donor transcripts identified. Host leader sequences snatched were generally between 16-20 nucleotides long. Most of the leader sequences ended up with AGA or GAG and it appeared that certain motifs were used more frequently. Analysis of the identified host donor transcripts revealed the abundant use of photosynthesis gene transcripts for TSWV cap-snatching. It was also found that heat stress (SG-induction) altered the usage of certain gene transcripts (Chapter 5). When functional GFP and non-functional GFP transcripts were simultaneously offered as cap donors in TSWV-infected plants both transcripts were used during TSWV cap-snatching. When a functional GFP transcript and a long GFP transcript were offered during a TSWV infection in planta, at normal and stress conditions, again both leaders were being used during cap-snatching. The results altogether indicated that TSWV was able to use transcripts destined to PBs and SGs, but also from possible upstream sources containing functional GFP transcripts (Chapter 5).
In summary, the research described in this thesis indicates that the cytoplasmic-replicating segmented NSVs likely apply a generic mode of cap-snatching with a potential role for P bodies and stress granules, but also the nucleopore complexes, as source and foci from where capped-donor RNAs may be used, as being discussed in Chapter 6. The abundant usage of transcripts from photosynthesis pathway genes during TSWV cap-snatching indicates that, although somewhat speculative, disease symptoms like chlorosis could at least in part, be the result of a specific translational shut-off by plant-infecting NSVs, resulting from cap-snatching from host mRNAs needed for proteins effective in photosynthesis. Further evidence, whether capped-mRNAs indeed are being used from the aforementioned granules/foci will have to come from future co-localization studies with the viral RdRp protein, the viral protein needed for endonuclease cleavage of leader sequences.
|Qualification||Doctor of Philosophy|
|Award date||6 Oct 2021|
|Place of Publication||Wageningen|
|Publication status||Published - 2021|
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- 1 Finished
A study into the roles of RNA granules for replication of segmented, negative strand plant RNA viruses in planta
Xu, M., Kormelink, R. & van Oers, M.
1/09/17 → 6/10/21