RNA-binding proteins of bovine rotavirus

Abstract

Two major bovine rotavirus proteins have RNA-binding activity as shown by an RNA overlay-protein blot assay. Of the six proteins in purified virions, only one showed RNA-binding activity. This 92,000-molecular-weight (92K) protein was present in both single- and double-shelled particles. Its RNA-binding activity was blocked by preincubation with monospecific antibody to VP2. Thus, the 92K RNA-binding protein in rotavirus virions is VP2, the second most abundant protein in single-shelled particles. In infected cell extracts, numerous cellular RNA-binding proteins and two virus-specific RNA-binding proteins were detected, VP2 and a 31K nonstructural (NS31) protein. VP2 bound single-stranded RNA in preference to double-stranded RNA, whereas NS31 bound both single- and double-stranded RNA equally well. Binding did not appear to be nucleotide sequence specific, because RNA from uninfected cells and an unrelated RNA virus bound to VP2 and to NS31 as did rotavirus RNA. This technique showed that both cellular and rotavirus RNA-binding proteins also bound DNA. VP2 interacted with rotavirus RNA over a broad pH range, with an optimum at pH 6.4 to 6.8, and at NaCl concentrations between 0 and 100 mM. The RNA-binding activity of NS31 exhibited similar pH and NaCl dependency. Sequence-specific nucleic acid binding could be detected by this method. When labeled synthetic oligodeoxyribonucleotides corresponding to the 3' and 5' plus-sense terminal sequences of rotavirus gene segments were used as probes, the 3' synthetic oligodeoxyribonucleotide bound to one 48K protein in control and infected cells. This suggests that there may be a specific functional interaction between the 48K cellular protein and this 3'-terminal noncoding region of the rotavirus genome or mRNA. These data show that the RNA overlay-protein blot assay is a useful test to identify some cellular and viral proteins with RNA-binding activity. For bovine rotavirus, the evidence suggests that, of all the virus-specific proteins, VP2 and NS31 are most likely to interact with RNA during transcription and replication or virus assembly or both.