Dokument

Summary:
Viruses in the Bunyaviridae family cause disease in humans ranging from a mild transient fever to viral haemorrhagic fever. The orthobunyavirus genus is the largest within the family and contains the La Crosse virus (LACV). LACV is endemic in the USA, causing 85 % of neuroinvasive viral disease in children under the age of 15. The main pathogenicity factor of LACV is the NSs protein, an inhibitor of the type I interferon (IFN) induction. Previous work in our group identified the mechanism of LACV NSs inhibition. During infection, the NSs protein of LACV induces the proteasomal degradation of the largest subunit, RPB1, in transcription elongating RNA polymerase II. As a possible host cell interactor of LACV NSs that could mediate the degradation of RPB1, was Elongin C identified. Elongin C has been described to have two main functions in the cell: 1) as a subunit of the Elongin complex that increases RNA polymerase II transcription elongation rates, and 2) as a subunit of several cellular and viral ubiquitin E3 ligases. Here, I demonstrate that LACV NSs specifically sequesters Elongin C from the nucleoli, but does not change the sub-cellular localization of the other two subunits of the Elongin complex, Elongin A and B. The LACV NSs re-localization of Elongin C from the nucleoli had minimal effects on the nucleolar structure or the localization of a major nucleolar protein, Nucleolin. The re-localization of Elongin C by LACV NSs could be prevented by inhibiting the main protein export factor of the nucleus, CRM1, but the same inhibition did not rescue RPB1 from degradation. However, siRNA mediated knockdown of Elongin C partially rescued RPB1 from degradation concomitantly with a partially rescued of type I IFN induction. In attempts to map the functional domains of LACV NSs, I was able to dissect the inhibition of general host cell transcription and type I IFN induction. All LACV NSs mutants, generated at conserved sites in the NSs protein throughout the orthobunyavirus genus, had lost the ability to inhibit type I IFN induction while they all retained the inhibition of general transcription. However, two of the mutants did not show robust phenotypes, requiring further studies to clarify their respective roles. For the rest of the mutants, the inhibition of general transcription correlated with RPB1 degradation, while the loss of type I IFN inhibition correlated partly with loss of Elongin C re-localization and/or inhibition of transcriptionally active RPB1. Thus, I have established that the re-localization of Elongin C by LACV NSs might play a role in type I IFN inhibition. Furthermore, I was able to dissect the inhibition of general host transcription and type I IFN induction transcription, pointing towards two different mechanisms of inhibition. General transcription is inhibited by RPB1 degradation, while type I IFN inhibition correlates partly with RNA polymerase II elongation inhibition.

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