Name
Characterisation of African Horse Sickness Virus’ non-structural protein 3 in insect and mammalian cells
Presenter
Juyeon Park, London School of Hygiene and Tropical Medicine
Co-Author(s)
Juyeon Park (LSHTM), Polly Roy (LSHTM)
Abstract Category
Virus Replication: Entry, Exit and Everything in Between
Abstract
African horse sickness virus (AHSV) is a member of the Orbivirus genus, and is the causative agent of economically significant African horse sickness. The viral nonstructural protein NS3 encoded by the smallest RNA segment 10 is known to play a key role in virus egress and cytotoxicity. However, the underlying mechanisms are poorly understood. My research aims to understand the cytotoxicity of NS3, and the interactions between NS3 and cytoplasmic proteins and to characterise its functions in both mammalian and insect cells using various biological and biochemical assays.
A loss of membrane integrity was observed when NS3 was expressed in the insect Sf9 and mammalian BSR cells, and this is particularly evident in the Sf9 cells, suggesting that AHSV NS3 can permeabilise the cell membrane, resulting in cell death. Since the cytotoxicity is serotype dependent, an AHSV1/4 reassortant virus was generated by replacing the S10 of AHSV1 with that of AHSV4 using reverse genetics. Wild-type AHSV1 displayed higher cytotoxicity compared to AHSV4 by showing larger-sized plaques. However, the reassortant virus showed an attenuated phenotype, indicating that NS3 is a determinant of virus cytotoxicity.
The interaction of host proteins with NS3 was also investigated. Cytoplasmic proteins Tsg101 and NEDD4 interacting with the late domain motifs of NS3 are involved in viral egress. Late-domain mutant viruses exhibited significant attenuation, suggesting a critical role of these motifs at later stages of the virus life cycle. Further studies utilizing mutant viruses and biochemical assays aim to elucidate the interactions between NS3 and intracellular proteins.
A loss of membrane integrity was observed when NS3 was expressed in the insect Sf9 and mammalian BSR cells, and this is particularly evident in the Sf9 cells, suggesting that AHSV NS3 can permeabilise the cell membrane, resulting in cell death. Since the cytotoxicity is serotype dependent, an AHSV1/4 reassortant virus was generated by replacing the S10 of AHSV1 with that of AHSV4 using reverse genetics. Wild-type AHSV1 displayed higher cytotoxicity compared to AHSV4 by showing larger-sized plaques. However, the reassortant virus showed an attenuated phenotype, indicating that NS3 is a determinant of virus cytotoxicity.
The interaction of host proteins with NS3 was also investigated. Cytoplasmic proteins Tsg101 and NEDD4 interacting with the late domain motifs of NS3 are involved in viral egress. Late-domain mutant viruses exhibited significant attenuation, suggesting a critical role of these motifs at later stages of the virus life cycle. Further studies utilizing mutant viruses and biochemical assays aim to elucidate the interactions between NS3 and intracellular proteins.