The poly(rC)-binding protein PCBP2 has multiple functions in post-transcriptional control of host and viral gene expression. Since it interacts with picornaviral RNA structures, it was proposed that PCBP2 regulates viral genome translation and replication. The hepatitis A virus (HAV), an atypical picornavirus, contains an unusual pyrimidine-rich tract (pY1) with unknown functions. Using in vivo and in vitro assays, we provide direct evidence that PCBP2 interacts with pY1 and that binding is mediated by KH domains 1 and 3. Proteolytic cleavage by the viral protease 3C generates a C-terminally truncated polypeptide with highly reduced RNA affinity. The results suggest that during HAV infection PCBP2 cleavage might specifically down-regulate viral protein synthesis, thereby giving way to viral RNA synthesis. (C) 2007 Elsevier Inc. All rights reserved.