Functional significance of the interaction of hepatitis A virus RNA with glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Opposing effects of GAPDH and polypyrimidine tract binding protein on internal ribosome entry site function

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involve d in glycolysis, binds specifically to several viral RNAs, but the function al significance of this interaction is uncertain. Both GAPDH and polypyrimi dine tract binding protein (PTB) bind to overlapping sites in stem-loop III a of the internal ribosome entry site (IRES) of Hepatitis A virus (HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary str ucture, we reasoned that GAPDH may suppress the ability of the IRES to dire ct cap-independent translation, making its effects antagonistic to the tran slation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. L emon, J, Biol. Chem. 271:14134-14142, 1996). To test this hypothesis, we co nstructed plasmids containing a dicistronic transcriptional unit in which t he HAV IRES was placed between an upstream GAPDH-coding sequence and a down stream Renilla luciferase (RLuc) sequence. Transfection with this plasmid r esults in overexpression of GAPDH and in RLuc production as a measure of IR ES activity. RLuc activity was compared with that from a control, null-expr ession plasmid that was identical except for a frameshift mutation within t he 5' GAPDH coding sequence. In transfection experiments, GAPDH overexpress ion significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells bu t not in Huh-7 cells, which have a significantly greater cytoplasmic abunda nce of PTB, GAPDH suppression of HAV translation was greater with the wild- type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P 16) that has reproducibly higher basal translational activity in BSC-1 cell s. Stem-loop ma RNA from the latter IRES had significantly lower affinity f or GAPDH in filter binding experiments, Thus, the binding of GAPDH to the I RES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells, The enhanced replication capacity of cell cultur e-adapted HAV in such cells may be due in part to reduced affinity of the v iral IRES for GAPDH.