The use of a reverse genetics system to identify the functional domains of NS2 during Bluetongue Virus replication

Bluetongue is a non-contagious arthropod transmitted viral infection of ruminants, especially sheep where mortality can reach up to 70%. Bluetongue Virus (BTV) has a genome of 10 double-stranded RNA segments which encode for 7 structural (VP1-7) and 4 non-structural proteins (NS1-4). A characteristic of BTV infected cells is viral inclusion bodies (VIBs), consisting mainly of NS2. The VIBs are the sites of immature virus assembly where the structural proteins VP3 and VP7, the transcription complex proteins (VP1, VP4, and VP6) and the BTV ssRNA are recruited and assembled. NS2 is a multifunctional protein which is thought to be essential during in vivo replication. Three putative ssRNA binding domains have been identified in NS2; mutations to these regions have been designed to disrupt ssRNA binding. Two systems have been used to study these mutations: a novel BTV reverse genetics system to rescue and characterise the effect of the mutations in vivo through analysis of virus growth, protein production and VIB formation; and a baculovirus protein expression system used to assess the structural changes of NS2, if any, oligomerisation and BTV ssRNA binding activity. This thesis presents the first in vivo and in vitro investigations into the ssRNA binding domains. The accumulated data demonstrated that mutation of the N-terminal domain had the most significant effect on BTV replication, its involvement in oligomerisation and BTV ssRNA binding. In contrast, the effect of the mutation of the middle and C-terminal RNA binding domains were less severe but still exhibited detectable differences in replication. In conclusion, NS2-ssRNA binding was shown to be essential for BTV replication.