Replication and Maturation of Viruses

Virus-Host Interactions

Opposed to viruses with a plus-strand RNA genome, viruses with a negative-strand, segmented RNA genome are only infectious and able to trigger a multiplication cycle in cells in the additional presence of the viral nucleoprotein (N) and the RNA-dependent RNA-polymerase (RdRp). Examples for these viruses can be found among many medically important viruses like Influenza virus (Orthomyxoviridae), Lasse fever (Arenaviridae) and Hantaan virus (Bunyaviridae), but also among plant viruses like Tomato spotted wilt virus (TSWV; Tospovirus genus within the Bunyaviridae).

All these viruses share several features on the structural and molecular level of which transcription initiation of the viral genome is the most unique one. During this process, referred to as cap-snatching, a nucleotide sequence between 10 and 20 nt in size is cleaved from the 5’ end of host mRNAs by an endonuclease activity encompassed within the viral RdRp. For Influenza this process occurs in the nucleus whereas for TSWV, and most other segmented (-)RNA viruses this happens in the cytoplasm. The capped leader obtained is subsequently used to prime transcription on the viral genome, which ultimately leads to the synthesis of capped, translatable viral mRNAs.

Model for TSWV cap-snatching
Model for TSWV cap-snatching

For TSWV it has been demonstrated that basepairing of cap-donor RNA leader sequences to the viral template RNA promote its use. For TSWV and Influenza A virus this process is currently being further investigated in vitro, using purified virus particles supplied with (mutant) cap-donor molecules to identify preferential cap-donor molecule sequences (topic 1).

 Proteins translated from viral transcripts will support the ongoing replication and subsequent virus maturation, ultimately leading to the accumulation of enveloped virus particles. For TSWV efforts are made to mimick this process, and eventually reconstitute viral RNPs and enveloped virus particles in mammalian cells (topic 2). TSWV S-RNA derived transcripts are characterized by the presence of a predicted hairpin structure at their 3’-ends, instead of a poly(A)-tail. The role of this hairpin structure in transcription/translation will be investigated in vitro and in vivo (topic 3). TSWV is being transmitted by thrips (Thysanoptera) in which the virus also replicates. Whereas the maturation pathway of TSWV in thrips seems to resemble those from the animal-infecting (bunya-) viruses in that enveloped virus particles eventually escape from the cell surface, TSWV particles accumulate and retain in large ER-derived vesicles in plant cells. As the viral membrane proteins Gn (G2) and Gc (G1) are the key mediators of virion assembly and release, the two glycoproteins  are transiently being expressed alone, together or in the additional presence of the structural N protein and subsequently studied in time for their localization behaviour in plant and mammalian cells (topic 4). Proteins-protein interactions required during intracellular trafficking are being analyzed by FRET/FLIM or bimolecular fluorescence complementation.  

TSWV is the type species of the tospoviruses, and ranks among the top 5 of economically most important plant viruses, causing great yield losses in many important crops like tomato, pepper, lettuce, groundnut and tobacco. At this moment more than 14 distinct tospovirus species have been identified and characterized, and one of the most recent ones is Tomato yellow ring virus (TYRV) from Iran. To estimate their economic importance and to allow for successful resistance breeding of various crops, as well as development of diagnostic tools (serology/ RT-PCR) new tospovirus isolates identified are characterized in terms of serology (using poly- and monoclonal antibodies), molecular properties, nucleotide sequence (divergence/phylogeny), host range and thrips vector choice (topic 5).

The research is embedded within the "Interactions between Plants and Biotic Agents" theme of the Graduate School Experimental Plant Sciences

Homodimerization of TSWV N protein visualized in mammalian cells by means of fluorescence lifetime imaging (FLIM) of CFP in a cell transfected with a: CFP-N, and b: CFP-N and YFP-N. A shorter fluorescence lifetime of CFP(-N) is clearly observed in the presence of YFP-N (panel b), as indicated by the pseudocolour change into yellow. The legend for the pseudocolours representing CFP fluorescence lifetime is provided in the colour scale on the right. The decrease in lifetime is a consequence of fluorescence energy transfer and thus of interaction between the fusion proteins.
Homodimerization of TSWV N protein visualized in mammalian cells by means of fluorescence lifetime imaging (FLIM) of CFP in a cell transfected with a: CFP-N, and b: CFP-N and YFP-N. A shorter fluorescence lifetime of CFP(-N) is clearly observed in the presence of YFP-N (panel b), as indicated by the pseudocolour change into yellow. The legend for the pseudocolours representing CFP fluorescence lifetime is provided in the colour scale on the right. The decrease in lifetime is a consequence of fluorescence energy transfer and thus of interaction between the fusion proteins.