African horsesickness virus (AHSV) is a member of the Orbivirus genus and part of the family Reoviridae. It is a double stranded RNA virus with ten genome segments that encode seven structural and four nonstructural proteins. AHSV nonstructural protein NS2, encoded by segment eight, is a single stranded RNA (ssRNA) binding protein. The formation of viral inclusion bodies (VIBs) as well as the selection and condensation of the ten different virus genome segments during encapsidation, are proposed functions of NS2. This study investigates the binding ability of NS2 to ssRNA by looking at the protein structure, as well as its affinity for virus specific mRNAs. NS2 has an α-helix rich C-terminal region and α-helixes are known to be involved in ssRNA binding. BTV NS2 has an α-helix content of 69% and AHSV NS2 an α-helix content of 47%. AHSV NS2 has a lower binding affinity for ssRNA than bluetongue virus (BTV) NS2. There thus seems to be a correlation between the α-helix content of different NS2 proteins and their ssRNA affinity. In order to determine if the difference between the ability of AHSV NS2 and BTV NS2 to bind nonspecifically to poly(U)Agarose can be ascribed to differences in the α-helix rich C-terminal of NS2, a chimeric NS2 protein was constructed that contained the α-helix rich C-terminal of BTV NS2 and the N-terminal of AHSV NS2. The binding affinity of the chimeric NS2 to poly(U)Agarose was compared to that of AHSV NS2 and BTV NS2 and it was found that it correlated well with that of AHSV NS2. The α-helixes therefore do not seem to play a major role in ssRNA binding. The binding affinity of NS2 rather seems to be determined by the N-terminal of the protein. In order to investigate the specific affinity of AHSV NS2 for AHSV mRNAs, it was necessary to obtain mRNA transcripts with terminal ends identical to AHSV mRNAs. The AHSV genes were cloned into a transcription vector with an internal ribozyme. The 3' ends of the transcripts were generated by autolytic cleavage mediated by the ribozyme immediately downstream of the viral insert. The vector's promoter was constructed in such a way that the 5' ends of the genes were inserted at the plus one position for transcription. Full-length mRNA transcripts of four AHSV genes were obtained, the genes encoding NS2, NS3, VP6 and VP7. mRNA transcripts of NS3 and VP7 genes, lacking three to five terminal nucleotides at either the 5' or 3' ends, were also obtained. A nonspecific mRNA control was derived from an AHSV gene cloned in the wrong transcriptional orientation. Preliminary binding assays showed that NS2 has at least a nonspecific affinity for all these mRNAs. The fluctuation in the results also suggests that mRNA concentration may playa role in protein/mRNA interactions. We suggest that NS2 may play a role in encapsidation by binding nonspecifically to all the mRNAs in the infected cell and thus presenting it at the VIBs for selection and encapsidation.
Dissertation (MSc (Genetics))--University of Pretoria, 2006.