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J. Biol. Chem., Vol. 279, Issue 36, 37822-37831, September 3, 2004

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Specific Interaction between Human Parechovirus Nonstructural 2A Protein and Viral RNA^*

Olga Samuilova, Camilla Krogerus, Tuija Pöyry¶, and Timo Hyypiä||

From the Department of Virology, Haartman Institute, University of Helsinki, P. O. Box 21, FIN-00014 Helsinki, Finland, the ^¶Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom, and the ^||Department of Medical Microbiology, University of Oulu, 90014 Oulu, Finland

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.

Received for publication, December 28, 2003 , and in revised form, June 23, 2004.

^* This work was supported by the Academy of Finland, The Sigrid Juselius Foundation, and the INTAS Project 011-2012. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Haartman Institute, Dept. of Virology, P. O. Box 21, FIN-00014 University of Helsinki, Helsinki, Finland. Tel.: 358-919126608; Fax: 358-919126491; E-mail: olga.samuildva{at}helsinki.fi.

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