Characterization of avian reovirus non structural protein σNS synthesized in Escherichia coli

The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein σNS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion protein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (eσNS) consistent with the expected molecular size of the avian reovirus protein σNS synthesized in infected cells was readily purified by His-Bind Resin. The eσNS was further confirmed to be indistinguishable from viral σNS by immunoblot analysis. The eσNS binds 32P-labeled ssRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding activity is blocked by heterologous yeast rRNA, but not by homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA-binding activity of the expressed protein σNS is non sequence-specific, similar to that previously described for viral σNS purified from avian reovirus infected cell extracts. In addition, the recent data also show that the optimal salt (NaCl) concentration and pH for its binding are 100–150 mM and 7.0, respectively, in terms of the UV cross-linking and RNase A treatment of the reaction mixtures prior to the denaturing gel analysis.