Cellular protein hnRNP-A1 interacts with the 3'-end and the intergenic sequence of mouse hepatitis virus negative-strand RNA to form a ribonucleoprotein complex.
We previously showed that several cellular proteins specifically bind to the 3'-end and the intergenic sequences of the negative-strand RNA of mouse hepatitis virus (MHV), and proposed that these distant RNA sequences can be brought together by cellular and viral proteins (Furuya and Lai, 1993; Zhang et al., 1994; Zhang and Lai, 1995). The cellular protein p35 has been identified as a heterogeneous nuclear ribonucleoprotein (hnRNP) A1. We have now expressed hnRNP-A1 as a glutathione-S-transferase (GST) fusion protein and demonstrated that the amino terminal two-thirds of hnRNP-A1 interacted with the two MHV regulatory RNA sequences (3'-end and intergenic sequences) through protein-RNA interaction while its carboxy-terminal glycine-rich domain mediated homomeric (protein-protein) interactions. In a partially reconstituted reaction, in which the two MHV RNA fragments and the purified GST-hnRNP-A1 fusion protein were mixed, an RNP complex was formed. Depletion of either hnRNP-A1 or one of the RNA components abolished the complex formation. These results indicate that hnRNP-A1 can mediate the formation of an MHV RNP complex, which includes both the negative-strand leader and intergenic sequences. Site-directed mutagenesis revealed that mutations in the MHV intergenic sequences, which inhibited MHV RNA transcription, also inhibited the RNP complex formation. Deletion analysis showed that the amino terminal RNA-binding domains of hnRNP-A1 is essential for the RNP complex formation while the carboxy-terminal protein-binding domain enhanced the complex formation by 90-fold. These findings provide direct evidence demonstrating that the negative-strand leader RNA and intergenic sequences can form an RNP complex mediated by cellular protein hnRNP-A1.
Zhang
X
X
Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Li
H P
HP
Xue
W
W
Lai
M M
MM
D000818:Animals; D001665:Binding Sites; D002874:Chromosome Mapping; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics*; D016296:Mutagenesis; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins