Abstract:
African horsesickness virus (AHSV) is a member of the Orbivirus genus within the Reoviridae family and causes an acute disease in horses. AHSV encodes four non-structural proteins (NS1, NS2, NS3/NS3A), whose functions in the viral life cycle are not fully understood. Amongst these, NS3 is believed to mediate virus release from infected cells. Data for bluetongue virus (BTV), the prototype orbivirus, has indicated that NS3 recruits cellular proteins to aid in the non-lytic release of virus particles. By making use of yeast two-hybrid screens, it was shown previously that the AHSV NS3 protein interacts with three insect cell proteins, namely Smad Anchor for Receptor Activation (SARA) protein, heat shock protein 70 (Hsp70) and ubiquitin (UB). Based on the involvement of host cell proteins in NS3-mediated non-lytic virus release, the aims of this study were thus to confirm the interactions between AHSV NS3 and the insect cell proteins in vitro and to determine whether these proteins may aid virus release from infected mammalian cells. To confirm the interaction of the AHSV NS3 protein with the insect cell proteins, the SARA, Hsp70 and UB peptides were expressed as glutathione S-transferase (GST)-tagged fusion proteins in E. coli. These GST fusion proteins were subsequently used in pull-down assays with E. coli lysates containing a truncated AHSV NS3 protein. No interaction between the virus and insect cell proteins could be demonstrated with this assay. To determine whether the SARA, Hsp70 and UB proteins may have biological relevance in the non-lytic release of AHSV from infected cells, a RNA interference (RNAi)-based approach was used. Pre-designed small interfering RNAs (siRNAs) that have been validated for silencing SARA, Hsp70 and UB gene expression in mice were evaluated for their ability to knockdown expression of their target genes in BHK-21 cells. The results indicated that mSARA-siRNA and mUB-siRNA down-regulated transcription of the SARA and UB genes by 63% and 56%, respectively, whereas the mHsp70-siRNA suppressed Hsp70 gene expression by only 3%. BHK-21 cells were subsequently transfected with the respective siRNAs in separate experiments followed by virus infection. Exposure of BHK-21 cells to these siRNAs did not result in a reduction in extracellular virus titres when compared to control cells. Although it is tempting to conclude that the SARA and UB proteins are not functionally relevant with regards to a role in AHSV egress, it is, however, also plausible that these results were due to the inability to reduce the level of target mRNA transcripts to such an extent that it would result in a loss of gene function. During the course of this study, various parameters that may have potentially influenced the results were identified. Further experiments are thus required before definitive conclusions can be drawn as to whether or not AHSV NS3 interacts with the respective insect cell proteins and, if so, what the relevance of these interactions might be with regards to virus release.
