Mapping the function of the NS3 protein of African horsesickness virus using RNAi and yeast two-hybrid analyses

Abstract

African horsesickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, has a ten-segment double-stranded (ds)RNA genome that is encapsidated within a single non-enveloped virus particle. In addition to seven structural proteins (VP1-VP7), four non-structural proteins (NS1, NS2 and NS3/NS3A) are synthesized in infected cells but their function in the viral life cycle is not yet fully understood. The non-structural protein NS3 is believed to be mediate virus release from infected cells. Although it has generally been accepted that non-enveloped viruses are released from infected cells after cellular lysis, available data for Bluetongue virus (BTV), the prototype orbivirus, has indicated that NS3 may form a bridge between the virus particle and the host cell budding machinery, and that it might have some controlling role in mature virus release from infected cells. Whether AHSV NS3 has a direct role in virus egress has not been addressed directly to date. Consequently, the aims of this investigation were to determine whether NS3 of AHSV has an intrinsic activity to release virus particles from infected cells and to determine whether NS3 may engage host cell proteins to aid virus release. To investigate whether AHSV NS3 is capable of mediating virus release, a RNA interference (RNAi)-based approach was used. Three small interfering RNAs (siRNAs) were designed that targeted different regions on AHSV-3 NS3 mRNA, i.e. siNS3-65 (corresponding to nucleotides 65-85), siNS3-74 (corresponding to nucleotides 74-92) and siNS3-266 (corresponding to nucleotides 266-284). Using a NS3 expression reporter plasmid and an in vitro model of infection, results were obtained that showed that the synthetic siRNAs, most notably siNS3-266, silenced NS3 mRNA and protein expression effectively. Moreover, silencing of NS3 gene expression with siNS3-266 resulted in a lower percentage of virus release when compared to control cells, albeit that the total virus yield was similar. These results thus provide evidence that AHSV release is enhanced by NS3. Towards defining host cell proteins that interact with the AHSV NS3 protein, a two-hybrid system was used that allows the detection of protein-protein interactions in vivo in yeast cells. Since AHSV infects both insect and mammalian hosts, cDNA libraries were constructed of Vero mammalian and Culicoides insect cell lines, and then screened for proteins interacting with the N-terminal of the AHSV-3 NS3 protein. As NS3-interacting cDNA clones, SARA was isolated from the insect cell cDNA library and L34 from the mammalian cDNA library. The SARA protein is localized to membranes and is implicated in membrane trafficking of proteins. The L34 protein is a multifunctional protein that, in addition to forming part of the 60S subunit of the ribosome, acts as an inhibitor of cyclin-dependent kinases 4 and 5, which play key regulatory roles during progression of the eukaryotic cell cycle. Moreover, L34 also regulates the synthesis of polyamines, which are involved in various cellular processes, including membrane stabilization. Although the biological significance of these interactions has yet to be investigated, the results nevertheless provide a foundation for mapping the role of NS3 in the AHSV life cycle.