Binding determinants in the interaction of the orbivirus non-structural protein NS2, with single-stranded RNA

Abstract

Morphogenesis of orbiviruses, of which bluetongue virus (BTV) is the prototype, entails a process whereby each precursor viral particle acquires a single copy of each of the ten dsRNA genome segments. The lack of free pools of dsRNA in infected cells has led to the assumption that the viral mRNA is packaged. Thus, ssRNA-binding proteins may play an important role in this process. Orbivirus mRNA bear 5' and 3' terminal hexanucleotide sequences which are common to all gene segments as well as short segment-specific inverted repeat sequences adjacent to the conserved terminal sequences. These conserved configurations provide circumstantial evidence for some functional role in encapsidation. Secondary structure analysis of BTV mRNA transcripts have predicted base-pairing at the inverted repeat sequences which results in 5' and 3' terminal overhangs as well as segment-specific stem-loop structures. It has previously been shown that the terminal hexanucleotide sequences are not required for optimal NS2-RNA binding. In this study, the importance of the inverted repeat sequences in RNA-protein binding was investigated. Using polymerase chain reaction (PCR) methodology, three different gene constructs lacking the 5' and/or 3' inverted repeat sequences, have been constructed. The PCR products were cloned into a transcription vector under control of the T7 RNA polymerase promoter. Cytoplasmic extracts from Spodoptera frugiperda cells infected with a baculovirus recombinant expressing the BTV NS2 protein and in vitro synthesized RNA transcripts were subjected to nitrocellulose RNA-protein binding assays. The cumulative results suggested that the inverted repeat sequences do not influence RNA-protein binding directly, but rather indirectly by altering the secondary structure of the RNA. Secondary structure motifs may thus mediate assortment of the viral mRNAs by acting as packaging signals for a specific RNA segment. BTV NS2 has a strong affinity for BTV transcripts, but it also binds to other single¬stranded RNA species. To investigate specificity in the RNA-NS2 interaction, an IAsys biosensor was used whereby the binding could be quantified. Transcription vectors containing segment 8 genes of different orbiviruses were constructed and the NS2 protein was expressed in a prokaryotic expression system as a GST fusion protein. No RNA-NS2 binding could be indicated. This approach may in future contribute to the study of RNA-protein binding interactions, if the problems as highlighted in this study, can be overcome. Computational secondary structure prediction of all ten BTV-10 mRNA transcripts, segment 8 transcripts of different orbiviruses and different mutant transcripts was also performed. Transcripts with intact 5' and 3' inverted repeat sequences all indicated base-pairing between the 5' and 3' ends. Secondary structure analysis of the mutant transcripts lacking the inverted repeat sequences indicated alterations in the secondary structures resulting from altered base-pairing. Conserved and unique secondary structures present on segment 8 mRNAs of different orbiviruses were identified, but their functional importance awaits further mutagenesis studies.