Analysis of tomato spotted wilt virus genome transcription

I.C. van Knippenberg

Research output: Thesisinternal PhD, WU

Abstract

Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus within the Bunyaviridae, a family of segmented negative strand RNA viruses. Although much ground has been covered in the past two decades, many questions concerning the mechanism of replication and transcription of this important group of viruses have thus far remained unanswered. Elucidation of the molecular mechanisms of viral transcription and replication requires manipulable systems in which these processes can be studied and unravelled. At the onset of the research described in this thesis, an in vitro assay was available in which RdRp (RNA-dependent RNA polymerase) activity ofpurified TSWV virions was observed, although it was unclear what kind of RNA synthetic activity was observed: transcription or replication.With the aim to unravel the molecular details of TSWV transcription, the in vitro assay was further developed into a well-defined transcription assay (Chapter 2). Transcription of TSWV, like that of all segmented negative strand RNA viruses, is initiated by cap snatching. In this process, a host mRNA (i.e. cap donor) is cleaved in its non-coding leader sequence by a virally encodedendonuclease,and the resulting capped leader is subsequently used to prime transcription of the viral genome. The existing in vitro system, in which only viral replication was observed, was modified by the addition ofrabbit reticulocyte lysate (RRL) which allowed viral transcription to take place. In this way a system was established in which the mechanism of cap snatching could be studied. Not only globin mRNAs present in the lysate, but also exogenously added capped RNA molecules were used as cap donors.The observed requirement for RRL seemed to imply a translational dependence of TSWV transcription. This possibility was further investigated in Chapter 3, using two inhibitors of protein synthesis. Surprisingly, in contrast to what has been observed for other Bunyaviruses, addition of these translation inhibitors had no effect on TSWV transcription in vitro. Moreover, no actual protein synthesis could be detected in the in vitro transcription assay, due to the assay conditions being incompatible with in vitro translation. These results indicated that TSWV transcription, unlike that of several other Bunyaviridae, is not coup led to translation.In the absence of RRL, viral replication was observed in the in vitro system (Chapter 2), although at a much lower rate than transcription. Not only RNPs (ribonucleoprotein complexes) as present in the lysed virus particles, but also purified cytoplasmic RNPs were active in RNA synthesis in the in vitro assay. However, in contrast to virion-RNPs, cytoplasmic RNPs were found to be highly active in replication (in the absence of RRL) but hardly active in transcription (in the presence of RRL). These findings imply the existence of RNPs in two different states: replication-mode and transcription-mode.As a first step towards understanding transcription termination, a rough mapping of the 3'-ends of the S RNA-encoded mRNAs was undertaken (Chapter 4). Viral mRNAs synthesised in the in vi/ra system were amplified by RT-PCR using primers for the leader sequence snatched from a-globin mRNAs in combination with a range of primers spanning the entire intergenic region (IR) of the S RNA segment. In addition, the size of the N mRNAs produced in infected plants was compared with the sizes of synthetic transcripts representing mRNAs terminated at defined positions in the intergenic region. The results indicated that transcription terminates near the 3'-end of the intergenic hairpin, yielding mRNAs that contain this hairpin structure at their 3' end, including a conserved sequence motif. These findings point toward a transcription termination mechanism reminiscent of that used by prokaryotes, where formation of a hairpin structure in the nascent transcript induces termination of transcription.Meanwhile, based on a concurrent PhD study, a model for TSWV cap snatching was proposed in which a cap donor (host mRNA leader) is required to have a single base complementarity to the viral temp late for transcription. The globin mRNAs in the RRL that are used as cap donor in the in vi/ra assay (Chapter 2) have 2 bases complementary to the viral temp late, which led to the question how far this complementarity could be extended. To investigate this, a capped transcript resembling a TSWV N mRNA, having a 15-nt stretch of complementarity, was tested and indeed found to be used as cap donor (Chapter 2), i.e. the 'viral mRNA' was re-snatched. The potential for re-snatching ofviral mRNAs occurring in vivo was investigated in Chapter 5. Competition assays using single-, double- and multiple-basepairing cap donors indicated a preference for donors with a long extent of base complementarity to the viral template. This implies that viral mRNAs would preferentially be used as cap donors, in other words re-snatching of viral mRNAs would prevail over snatching of host mRNAs. In addition, primer extension analyses on the products of a re-snatching reaction were used to examine the exact endonuclease cleavage site of re-snatching, indicating that endonuclease cleavage takes place after the first or second nucleotide complementary to the viral template. In chapter 6 the results presented in chapters 2-5 and their implications are discussed in relation to current knowledge on replication, transcription, and translation of negative and ambisense RNA viruses. The current model for the mechanism of cap snatching is fine-tuned further in view ofthe findings reported in Chapter 5.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Kormelink, Richard, Co-promotor
  • Goldbach, R.W., Promotor, External person
Award date16 Feb 2005
Place of PublicationWageningen
Publisher
Print ISBNs9789085040880
Publication statusPublished - 16 Feb 2005

Keywords

  • tomato spotted wilt virus
  • plant viruses
  • genomes
  • transcription

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