Characterisation of the African horse sickness virus NS4 protein

Jin, Yi (2018) Characterisation of the African horse sickness virus NS4 protein. PhD thesis, University of Glasgow.

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African horse sickness is one of the most deadly infectious diseases of horses. The disease is caused by African horse sickness virus (AHSV), an arbovirus transmitted by culicoides midges. AHSV is classified within the genus Orbivirus, family Reovirdae. The AHSV genome is composed by ten segments of double stranded RNA (dsRNA) encoding seven structural and at least four non-structural (NS) proteins.
AHSV shares structural and functional similarities with Bluetongue virus, the ‘prototype’ species of the genus Orbivirus. An alternative open reading frame (ORF), overlapping the main ORF encoding the VP6, has been identified in segment 9 in both AHSV and BTV. This additional ORF encodes the non-structural protein NS4. The BTV NS4 localises in the nucleoli of the infected cells. NS4 is an interferon antagonist and a determinant of virus virulence.
In this thesis, I aimed to characterise the AHSV NS4. Unlike the BTV NS4, the AHSV NS4 are relatively variable mong different virus strains. I have divided these proteins into four different subtypes: NS4I, NS4-IIα, -IIβ, and IIγ based upon their sequence similarity and on the presence of N-terminal or C-terminal truncations. In contrast to BTV, all four of these NS4 types localise in the cytoplasm of either transfected or infected cells. In addition, in transient transfection assays all the NS4 types show the ability to hamper gene expression, with NS4-IIβ being the most efficient.
In order to further understand the biological significance of the AHSV NS4 we used reverse genetics to generate viruses expressing the four types of NS4 (AHSV-NS4-I, AHSV-NS4-IIα, AHSV-NS4-IIβ, AHSV-NS4-IIγ) and the corresponding NS4 deletion mutants (AHSV-ΔNS4-I etc.).
Deletion of NS4 did not affect virus replication kinetics in either KC cells or interferon incompetent cells such as the BSR cell line. Similarly, both AHSV-NS4-IIβ and the corresponding ΔNS4 mutant showed similar replication kinetics in the interferon competent E. Derm cell line and in primary horse endothelial cells. In contrast, AHSV-NS4-I, AHSV-NS4-IIα, and AHSV-NS4-IIγ replicated more efficiently than the corresponding ΔNS4 viruses in these horse cells. Interestingly, the defects in replication of the NS4 viruses were removed after treatment with an inhibitor of the JAK/STAT pathway. Indeed, we observed that primary horse cells infected with the NS4 mutants released higher levels of type I interferon (IFN) than cells infected with the corresponding NS4 expressing viruses. In addition, we found the NS4 to be a determinant of virus virulence in vivo in NIH-Swiss mice infected with the viruses described above.
Collectively, the data described in this thesis suggest that the NS4 is one of the proteins used by AHSV to modulate the IFN response.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: AHSV NS4 IFN antagonist.
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Supervisor's Name: Palmarini, Prof. Massimo and Stewart, Dr. Meredith
Date of Award: 2018
Depositing User: Mr Yi JIN
Unique ID: glathesis:2018-8973
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Apr 2018 09:58
Last Modified: 01 Aug 2022 08:42

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