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Title: Interactions of Bunyamwera virus nucleocapsid protein and encapsidation of viral RNA
Author: Osborne, Jane C.
ISNI:       0000 0001 3460 4628
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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This project concerns the identification and characterisation of some of the molecular interactions of the Bunyamwera virus (BUN) nucleocapsid (N) protein, and attempts to construct a model for encapsidation of viral RNA by N. BUN is the prototype virus of the Bunyaviridae, a family of negative-strand viruses with tripartite genomes. All BUN genome and antigenome RNAs are encapsidated by N. This interaction was investigated in vitro by expressing His-tagged BUN N in bacteria, purifying it in its native form and developing binding assays to analyse its association with a short radiolabelled riboprobe consisting of the termini of the BUNS segment. N was demonstrated to bind the riboprobe by Northwestern, gel electrophoretic mobility shift (GEMSA) and filter-binding assays. The complexes were found to possess a similar level of resistance to digestion with ribonuclease as authentic nucleocapsids. Analysis by GEMSA was interpreted to indicate complete encapsidation of the riboprobe by N, with a number of discrete complexes presumed to be intermediates in the sequential encapsidation process apparent. Filter-binding assays were utilised to determine the binding kinetics. The resultant dissociation constant was similar to dissociation constants obtained for other negative-strand virus N-RNA interactions and implied that binding was strong. Supporting the latter observation was the ability of complexes to form over a wide range of ionic conditions. The binding kinetics also indicated that the binding of N to the riboprobe was co-operative, reinforced by the demonstration that the capacity of N to bind RNA was dependent on its concentration. The 5' terminus of each segment RNA had been implicated in encapsidation initiation, but no direct evidence had been produced. To investigate the presence of an encapsidation signal, competitive binding assays were set up with various RNA species. The 5' 32 terminal nucleotides of the BUN S segment were bound selectively, implicating this region in encapsidation initiation. In addition, N was capable of binding any RNA non-selectively and to a low degree, indicating the presence of two modes of binding. Predictions of the secondary structure of 5' terminal sequences revealed potential stem-loops containing a consensus sequence in the loop region that had previously been found to be essential for transcription of a recombinant BUN S segment in a minireplicon system. The stem-loop was suggested to constitute an encapsidation signal, supported by the inability of an RNA containing the same sequence but not predicted to form the stem-loop to be bound selectively. BUN mRNA is not normally encapsidated and possesses a capped, heterogeneous primer sequence on its 5' end. The predictions of secondary structure were extended to propose a mechanism of inhibition of encapsidation by the primer sequence, which, under certain circumstances, was suggested to be reversible when required. The information obtained on N-RNA interactions was used to propose a model for encapsidation in BUN. The co-operative nature of N-RNA binding suggested that multimerisation of N took place. This was investigated by expressing N as fusion proteins in the mammalian two- hybrid system, and a potential self-association was identified. This was supported by the co-immunoprecipitation of native N with His-tagged N in mammalian cells. However, neither the amino nor the carboxy half of N was found to be capable of interacting with His-tagged N exclusively. No evidence of an interaction between the viral polymerase, L, and N or between L and the non-structural viral protein NSs was obtained using the mammalian two-hybrid system, and NSs was not found to multimerise. However, a weak interaction between N and NSs was identified. The potential role of this interaction in the mechanisms of transcription inhibition or interferon antagonism ascribed to NSs is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Bunyaviridae; Molecular; Negative strand; Genomes