Specific interaction between human parechovirus nonstructural 2A protein and viral RNA

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.