The 3'-untranslated region (3'-UTR) of mouse hepatitis virus (MHV) RNA regulates the replication of and transcription from the viral RNA. Several host cell proteins have previously been shown to interact with this regulatory region. By immunoprecipitation of UV-cross-linked cellular proteins and in vitro binding of the recombinant protein, we have identified the major RNA-binding protein species as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). A strong hnRNP A1-binding site was located 90 to 170 nucleotides from the 3' end of MHV RNA, and a weak binding site was mapped at nucleotides 260 to 350 from the 3' end. These binding sites are complementary to the sites on the negative-strand RNA that bind another cellular protein, polypyrimidine tract-binding protein (PTB). Mutations that affect PTB binding to the negative strand of the 3'-UTR also inhibited hnRNP A1 binding on the positive strand, indicating a possible relationship between these two proteins. Defective-interfering RNAs containing a mutated hnRNP A1-binding site have reduced RNA transcription and replication activities. Furthermore, hnRNP A1 and PTB, both of which also bind to the complementary strands at the 5' end of MHV RNA, together mediate the formation of an RNP complex involving the 5'- and 3'-end fragments of MHV RNA in vitro. These studies suggest that hnRNP A1-PTB interactions provide a molecular mechanism for potential 5'-3' cross talks in MHV RNA, which may be important for RNA replication and transcription.