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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

  • Xuming Zhang
  • Hsin-Pai Li
  • Wenmei Xue
  • Michael M. C. Lai
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 440)

Abstract

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 vi-ral 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) Al. We have now expressed hnRNP-Al as a glutathione-S-transferase (GST) fusion protein and demonstrated that the amino terminal two-thirds of hnRNP-Al 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-Al fusion protein were mixed, an RNP complex was formed. Depletion of either hnRNP-Al or one of the RNA components abolished the complex formation. These results indicate that hnRNP-Al can mediate the formation of an MHV RNP complex, which includes both the negative-strand leader and intergenic se-quences. Site-directed mutagenesis revealed that mutations in the MHV intergenic se-quences, which inhibiterd MHV RNA transcription, also inhibited the RNP complex forma-tion. 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.

Keywords

Intergenic Sequence Heterogeneous Nuclear Ribonucleoprotein Mouse Hepatitis Virus Reconstitution Reaction Vitro Formation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Xuming Zhang
    • 1
    • 4
  • Hsin-Pai Li
    • 2
  • Wenmei Xue
    • 2
  • Michael M. C. Lai
    • 1
    • 2
    • 3
  1. 1.Department of NeurologyUniversity of Southern California School of MedicineLos AngelesUSA
  2. 2.Department of Molecular Microbiology and ImmunologyUniversity of Southern California School of MedicineLos AngelesUSA
  3. 3.Howard Hughes Medical InstituteUniversity of Southern California School of MedicineLos AngelesUSA
  4. 4.Department of Microbiology and ImmunologyUniversity of Arkansas for Medical SciencesLittle RockUSA

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