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Structural Analyses of Borna Disease Virus Nucleoprotein- Phosphoprotein and Nucleoprotein- RNA Interactions
Borna disease virus (BDV) is the only representative of the Bornaviridae in the order Mononegavirales. It is unique among the animal viruses of this order with respect to its transcription and replication in the nucleus, which provides access to the splicing machinery. BDV is noncytolytic, highly ne...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2009
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| Online Zugang: | PDF-Volltext |
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| Zusammenfassung: | Borna disease virus (BDV) is the only representative of the Bornaviridae in the order Mononegavirales. It is unique among the animal viruses of this order with respect to its transcription and replication in the nucleus, which provides access to the splicing machinery. BDV is noncytolytic, highly neurotropic and causes diseases of the central nervous system (CNS) in a wide range of vertebrates. As in other Mononegavirales, the BDV polymerase complex or ribonucleoprotein complex, consists of the nucleoprotein N, the phosphoprotein P, the polymerase L and viral genomic RNA. In the case of BDV another protein is involved, termed protein X.
BVD N forms a homotetramer and does not spontaneously interact with cellular RNA. Each protomer consists of two helical domains and N- and C-terminal extensions, involved in domain exchange and tetramer stabilization.
An open question remained how BVD N interacts with RNA, although overall structural similarities with nucleoproteins from rhabdoviruses and vesiculoviruses suggested similar modes of RNA interaction.
Protein P plays an essential role in assembly and regulation of the polymerase complex via interactions with X, N, L and itself. Oligomerization of P is required for the formation of an active polymerase complex, similar to other negative strand RNA polymerase complexes.
P requires an intact C-terminus for N interaction and may contact two different sites on N. Phosphoproteins from Rhabdoviruses and Sendai virus contain two different binding sites for N, one to keep N soluble and free from unspecific RNA and the other to bind to N-RNA complexes forming the polymerase complex together with the polymerase L. However, BVD N does not require P binding to prevent non-specific RNA interaction, since BDV N oligomerizes spontaneously into tetramers that do not complex RNA, thus the precise role of N-P interaction in the absence of RNA is not known.
The aim of our study was to understand the interaction between the BDV nucleo- and the phosphoprotein as well as the nucleoprotein and the viral RNA. Even though, no conclusive data were obtained upon crystallographic approaches, concerning N in complex with different truncated P-constructs and BDV genomic RNA, we present data about N-P and N-RNA interactions.
I show that P’, an N-terminally truncated isoform of the phosphoprotein, present in BDV infected cells, oligomerizes into tetramers. The tetrameric P’ interacts with BDV-N, thus forming hetero-octamers. The P’-N interaction requires five C-terminal amino acids of P’ to form a stable complex with a kD of 1.66 μM.
Tetrameric N is destabilized in the presence of 5’ genomic BDV RNA, which leads to the formation of N-RNA polymers. Similar N-RNA polymers are formed in the presence of P’, leading to P’-N-RNA polymers. Electron microscopy analyses of N-RNA and N-P’-RNA complexes revealed large “open” ring-like and string-like assemblies with the RNA exposed and accessible for degradation. The N or N-P polymers remain intact after RNA degradation indicating that polymerization is not mainly stabilized by RNA interaction. The N-RNA interaction is mediated via recognition of basic residues within the cleft of the N-and C-terminal domains similar to the observed nucleoprotein-RNA recognition of other negative strand-RNA viruses.
In conclusion, these data provide insight on the molecular interactions between the viral RNA and the nucleo- and phosphoprotein of the BDV ribonucleoprotein complex. |
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| Umfang: | 115 Seiten |
| DOI: | 10.17192/z2010.0095 |