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

Hepatitis A virus (HAV) is a plus-strand RNA virus that is classified within the genus Hepatovirus of the family Picornaviridae. The genomic RNA of HAV contains a single long open reading frame encoding a polyprotein which is proteolytically processed by a virus-encoded proteinase (27). Like other picornavirus RNAs, it has a relatively lengthy 5′-terminal nontranslated RNA segment (5′NTR) and a shorter 3′NTR with a 3′ terminal poly(A) tail. As in all picornaviruses, the 5′NTR of HAV forms a highly ordered RNA structural element (the internal ribosome entry site [IRES]) that is responsible for directing the cap-independent translation of the polyprotein (5, 23, 32). This occurs by a unique mechanism that involves internal entry of the ribosome on the RNA hundreds of bases downstream of the 5′ end and is absolutely dependent on the secondary and probably the tertiary structure of the 5′NTR. Although HAV can be propagated in conventional cell culture systems, the replication of wild-type (wt) virus is slow and leads to persistent infection in most cases without a cytopathic effect (1, 9, 35). However, cell culture adaptation occurs as the result of continued passage of virus (7, 19, 24). Generally, it is accompanied by more efficient replication and increased yields of virus. Several cell culture-adapted HAV variants are attenuated for their ability to induce liver injury in primates, although cell culture adaptation and attenuation are distinct phenotypes (29, 40, 42). The genomes of several cell culture-adapted HAV strains have been completely sequenced (7, 18, 24). One of these cell culture-adapted viruses, HM175/P16, differs from its wt parent, HM175/wt, by only 19 nucleotide (nt) substitutions scattered throughout the entire genome (24). In general, mutations within the P2 region of the genome (nonstructural proteins) and the 5′NTR of cell culture-adapted viruses are important in enhancing the capacity of these viruses to replicate in cultured primate cells (11, 13, 15). In particular, mutations within the 5′NTR of HM175/P16 (at nt 152, 203 to 204, and 687) confer cell-type-specific enhancement in viral translation and replication (10, 11, 37). A number of studies suggest that efficient translation by IRES elements requires not only canonical translation initiation factors but also noncanonical host factors (2–4, 17, 20, 22, 28, 31). Therefore, it is likely that these 5′NTR mutations enhance translation by altering the affinity of the RNA for cellular proteins that either positively or negatively influence the activity of the IRES. In previous studies, we identified cellular proteins of 30, 39, 57, and 110 kDa (p30, p39, p57, and p110) which interact specifically with RNA segments within the HAV IRES (6). p39 was the dominant RNA binding protein present in the ribosomal salt wash fraction (RSW) of HAV-permissive BSC-1 cells (African green monkey kidney cells) and FrhK-4 cells (fetal rhesus kidney cells). In contrast, p57 rather than p39 was the dominant RNA binding protein in rabbit reticulocyte lysates as well as RSW prepared from HeLa cells and Huh-7 (human hepatocellular carcinoma) cells (6; K. H. Chang, W. Klymstra, and S. M. Lemon, unpublished data). p57 was shown to be polypyrimidine tract binding protein (PTB), since it reacted with anti-PTB antibody in an immunoblot analysis (6), while the p39 protein was subsequently purified and identified as the cellular glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (36). GAPDH and PTB compete with each other for binding to overlapping sites on this RNA segment, which forms the 5′-most RNA structure in the HAV IRES (5). GAPDH has generally been considered to be a housekeeping protein involved mainly in glycolysis, but a number of recent studies indicate that it is multifunctional, possibly also playing roles in endocytosis, DNA replication, DNA repair, and RNA transport and/or translation (39). GAPDH specifically binds several cellular RNAs including the AU-rich 3′NTR of human lymphokine mRNA, as well as human tRNA (30, 38). In addition, after our demonstration of the specific binding of GAPDH to the 5′NTR of HAV, the enzyme was shown to bind specifically to 3′NTR sequences of human parainfluenza virus and hepatitis C virus as well as the RNA pregenome of hepatitis B virus (12, 34, 45). All of these RNAs appear to bind to GAPDH within the NAD+ binding groove of the enzyme (30, 36). Using circular dichroism spectropolarimetry, we found that the binding of GAPDH to stem-loop IIIa RNA of HAV resulted in significant destabilization of RNA secondary structure, suggesting that this interaction would be detrimental to IRES-directed translation (36). However, the functional impact of the binding of GAPDH to the HAV IRES has been difficult to demonstrate due to the ubiquitous nature and high constitutive expression levels of the enzyme. Stem-loop IIIa of the cell culture-adapted HM175/P16 virus contains a 2-nt UU deletion within a 5-nt oligo(U) tract in the wt virus (nt 200 to 204), which contributes to both the enhanced IRES activity and greater replication capacity that characterizes this virus in BSC-1 cells (11, 37). GAPDH preferentially binds U-rich segments of RNA (31, 37), making it likely that this mutation reduces the affinity of this RNA segment for GAPDH. Since GAPDH is the dominant protein in lysates of BSC-1 cells that is bound by this stem-loop of the IRES (36), we have reasoned that this mutation may be selected during the adaptation of the virus to growth in BSC-1 cells because it protects the IRES against the structure-destabilizing effects of GAPDH and thus promotes both cap-independent translation and, secondarily, viral replication. Here, we report experiments that provide insight into how the binding of GAPDH to HAV RNA influences IRES function and how this phenomenon might relate to mutations that are selected for within the IRES during the adaptation of HAV to growth in cultured cells. We constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and downstream Renilla luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. As a control, cells were transfected in parallel with a null-expression plasmid that was identical except for a frameshift mutation within the 5′ GAPDH coding sequence. The results of these experiments indicate that GAPDH overexpression significantly suppresses the ability of the HAV 5′NTR to direct cap-independent translation in transfected cells. We also demonstrate that the mutation at nt 203 to 204 in the cell culture-adapted virus specifically reduces the affinity of stem-loop IIIa for GAPDH, supporting the hypothesis that this mutation was selected because it reduces the negative impact of GAPDH on IRES-dependent translation and viral replication.