Analysis of the transcription initiation mechanism of tomato spotted wilt virus

Genome replication and transcription of Tomato spotted wilt virus (TSWV, genus Tospovirus ) follows in most aspects the general rules for negative strand RNA viruses with segmented genomes. One common feature is the occurrence of "cap snatching" during transcription initiation. During this process, capped leader sequences of suitable host mRNAs are recruited and cleaved to serve as primers for transcription.At the start of the research project described in this thesis, the occurrence of cap snatching during transcription initiation of TSWV was established, but the requirements for host leaders to function as cap donors were unknown. In order to identify these requirements under conditions that resembled a natural infection most closely, Alfalfa mosaic virus (AMV) RNAs were presented and tested as specific cap donors during a co-infection with TSWV of Nicotiana benthamiana (Chapter 2). Thus it was found that all four AMV RNAs can be used by TSWV as cap donors. Of these AMV RNAs, only RNA 3 was cleaved exclusively at one position, i.e. at nucleotide A17, indicating that a cleavage preference existed. However, as the 5' ultimate residue of the TSWV genomic sequence in these mRNAs could be either derived from the AMV leader or represent the first templated nucleotide, the actual cleavage site, i.e. 5' or 3' of the A17 residue, could not be established. In case the first viral residue originated from the capped leader, it would implicate a requirement for a single base pairing between the cap donor RNA and the viral template during transcription initiation.To confirm whether such single complementarity was required, a series of AMV RNA3 and RNA4 mutants, modified in their leader sequences, was offered as cDNA constructs to transgenic N. tabacum p12 plants expressing the AMV replicase proteins and tested as cap donors during a co-infection with TSWV. RT-PCR amplification and sequence analyses of chimaeric AMV-TSWV mRNAs strongly supported a single base pairing requirement between cap donor and viral template and a cleavage preference for nucleotide position 16 from the cap structure. Base pairing not only occurred with the 3' ultimate residue of the viral template, but in some cases also with the penultimate or even antepenultimate residues, indicating that a G-C base pairing instead of an A-U base pairing could also occur. These findings were further tested and substantiated by investigating TSWV mRNAs with leaders derived from known host mRNAs (Chapter 3).While the co-inoculation approaches using (mutant) AMV RNAs allowed the analysis of cap donor requirements for transcription initiation on TSWV genomic RNA, it was not suitable for the identification and characterisation of the viral proteins involved, such as the nucleoprotein (N) and RNA-dependent RNA polymerase (RdRp), or of important cis -acting sequences within the viral RNA template. Therefore, a Vaccinia virus -T7 expression system was developed, which enabled reconstitution of active viral RNPs entirely from cloned cDNAs (Chapter 4). Using this system, the 331.5 kDa RdRp was successfully expressed in OST7-1 murine fibroblasts along with the N protein and a reporter construct resembling the viral S RNA. By replacing the S RNA-encoded NSs gene for the sensitive luciferase reporter gene, functional RdRp activity was demonstrated with the expression of luciferase in vivo .In Chapter 5, the results obtained are discussed in relation to available data for other segmented negative strand RNA viruses. Finally, a model for TSWV transcription initiation is proposed.