Structure and expression of the tomato spotted wilt virus genome : a plant-infecting bunyavirus

This thesis describes studies which are aimed at the elucidation of the genetic organisation and expression strategy of the tomato spotted wilt virus (TSWV) RNA genome.

Using specific cDNA clones, corresponding to all three genomic RNA segments, the synthesis of virus specific RNA species in systemically infected Nicotiana rustica plants was followed (Chapter 2). These analyses revealed the presence of low (steady state) levels of vc strands and accumulating amounts of v strands, and confirmed that TSWV replicates as a negative strand RNA virus. Moreover, it could be demonstrated that the two genes contained in the S RNA are transcribed from complementary strands, confirming the ambisense nature of this genome segment.

To gain more information on the first steps in the transcription process of the TSWV genome, the subgenomic mRNAs, transcribed from the S RNA, were partially purified from infected plant material and their 5' ends analysed by primer extension. These analyses revealed the presence of non-viral, heterogeneous sequences, 12-20 nucleotides in length, at the 5' end of the mRNAs (Chapter 3). These observations indicated that the initiation of transcription of the viral genome most likely occurs via a mechanism referred to as "cap-snatching", a process that was first described for influenza virus.

The elucidation of the nucleotide sequence of the M RNA, as described in Chapter 4, revealed an ambisense coding strategy for this RNA segment. Meanwhile, TSWV had been classified as the representative of a newly created genus, denoted Tospovirus , within the Bunyaviridae, a family that until then only consisted of animal-infecting viruses. Thus it was concluded that TSWV represents a bunyavirus with two ambisense RNA segments. In M RNA a large open reading frame (ORF) was localized on the viral complementary (vc) strand, encoding the precursor to both envelope glycoproteins, G1 (78 kDa) and G2 (58 kDa). The second, smaller ORF, located on the viral strand of M RNA, encoded a putative nonstructural protein, denoted NS _M , of 33.6 kDa. The amino acid sequence of the glycoprotein precursor revealed the presence of an RGD-motif in the N-terminal region of G2. It was therefore anticipated that the glycoproteins are involved in acquisition and transmission by thrips via receptor binding.

Comparison with the genomes of the animal-infecting members of the Bunyaviridae, reveals that the second ORF within the M RNA of TSWV, i.e. the NS _M gene, represents an addition to the standard bunyaviral genome, suggesting that this gene might be involved in the adaptation of this bunyavirus to plant hosts. To gain more insight into the function of the nonstructural proteins of TSWV, both the NS _S and NS _M genes were cloned and expressed in heterologous expression systems. The proteins thus produced were purified and used for the production of polyclonal antisera. In Chapter 5, immunogold labelling analyses demonstrated the presence of the NS _S protein in fibrous structures within the cytoplasm of infected cells. Depending on the isolate, these structures were arranged in flexible or paracrystalline arrays. Similar studies were performed for the NS _M protein (Chapter 6), and revealed a transient character of this protein in a time course analysis of systemically infected plants. Immunogold analyses showed the association of NS _M with both non- enveloped nucleocapsid aggregates and plasmodesmata, though only during a short period early after infection. These results provided evidence that NS _M represents the viral movement protein, involved in cell-to-cell transport of non-enveloped ribonucleocapsids.

As a first step towards unravelling the maturation pathways of the glycoproteins, the gene for the glycoprotein precursor was cloned and expressed in the eukaryotic baculovirus/insect cell expression system. The results obtained, and presented in Chapter 7, demonstrated the actual glycosylation of the viral glycoproteins. Moreover, the precursor to the glycoprotein was found to be glycosylated, indicating that glycosylation takes place at the stage of the non-processed G1/G2 precursor protein. Finally, in Chapter 8 a general discussion is presented within the family Bunyaviridae, The evolutionary relationship to the animal-infecting members of the Bunyaviridae, is emphasized.