Specific Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase with the 5′-Nontranslated RNA of Hepatitis A Virus

doi:10.1074/jbc.271.24.14134

Specific Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase with the 5′-Nontranslated RNA of Hepatitis A Virus*

From the ^‡ Departments of Medicine and Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030 and the
^§ Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

^¶ To whom correspondence should be addressed: Tel.: 919-966-2536; Fax: 919-966-6714.

Abstract

Initiation of translation of hepatitis A virus (HAV) RNA occurs by internal entry and is likely to involve the interaction of trans-acting cellular protein factors with cis-acting structural elements of an internal ribosomal entry segment (IRES) within the 5′-nontranslated RNA. To characterize interactions between African green monkey kidney (BS-C-1) cell proteins and the predicted stem-loop IIIa (nucleotides 155–235) located at the 5′ border of the HAV IRES, we utilized an electrophoresis mobility shift assay (EMSA) to identify a 39-kDa RNA-binding protein (p39). Amino-terminal amino acid sequencing of highly purified p39 revealed absolute identity with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The identity of p39 as simian GAPDH was further confirmed by antigenic and biochemical similarities between p39 and human GAPDH. Analysis of the RNA binding properties of simian GAPDH revealed that this cellular protein interacts with two additional sites in the HAV 5′-nontranslated RNA, one located between nucleotides 1–148 and the other between nucleotides 597–746. Competitive EMSAs also demonstrated that GAPDH and human polypyrimidine tract-binding protein, a putative picornavirus translation initiation factor, compete with each other for binding to stem-loop IIIa, suggesting that the relative cytoplasmic abundance of GAPDH and polypyrimidine tract-binding protein in individual cell-types may be an important determinant of viral translation activity. Human GAPDH was found to destabilize the folded structure of the stem-loop IIIa RNA based upon observed decreases in the circular dichroism spectra of this RNA following binding of the protein. This RNA helix-destabilizing activity of GAPDH could directly influence IRES-dependent translation and/or replication of picornavirus RNA.

Footnotes

↵* This work was supported by Grants RO1-AI32599, T32-AI07151, and GM47431 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Ki Ha Chang, Ph.D., who first recognized the viral RNA binding activities of the p39 protein.
↵¹ The abbreviations used are:

HAV

hepatitis A virus

nt

nucleotide(s)

IRES

internal ribosomal entry segment

RSW

ribosomal salt wash

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

mAb

monoclonal antibody

EMSA

electrophoresis mobility shift assay

PAGE

polyacrylamide gel electrophoresis

PTB

polypyrimidine tract-binding protein

NTR

nontranslated region.
↵² S. F. Chao, R. E. Lanford, and S. M. Lemon, unpublished results.
↵³ D. E. Schultz, and S. M. Lemon, unpublished results.
- Received January 18, 1996.
- Revision received March 27, 1996.