These studies are aimed at characterizing structure-function relationships within the 5 nontranslated RNA (5NTR) of hepatitis A virus (HAV). A 39 kDa protein which binds specifically to parts of the HAV 5 NTR was identified as glyceraldehyde 3 phosphate dehydrogenase (GAPDH). GAPDH binding destabilizes stem-loop IIIa of the viral internal ribosomal entry sequence (IRES). GAPDH and pyrimidine tract binding protein (PTB), a cellular protein which facilitates HAV translation, compete with each other for binding to HAV RNA.
Specific aim 1 will determine: (1) whether mutations which enhance translation in BS-C-1 cells reduce the affinity of the 5NTR for GAPDH, thereby limiting GAPDH-mediated perturbation of RNA structure; (2) whether binding of GAPDH to the IRES destabilizes RNA structures other than stem-loop IIIa; and (3) whether binding of PTB also destabilizes structures within the IRES and, if not, whether PTB binding protects RNA structures from destabilization by GAPDH.
Specific aim 2 addresses the functional significance of GAPDH binding to the 5 NTR and will assess (1) effects on HAV translation with depletion of GAPDH in reticulocyte lysates, and (2) effects on viral translation and replication associated with modulation of GAPDH expression in vivo.
Specific aim 3 is to determine the identity of a 30 kDa cellular protein which binds to the IRES, and to assess its functional roles in viral translation and replication.
Specific aim 4 focuses on a novel series of temperature-sensitive (ts) HAVs with large deletion mutations involving the 5 pyrimidine-rich tract (pY1) and an adjacent 3 single-stranded RNA domain (nts 140-144), and includes (1) biophysical analysis of the higher ordered structure of the pY1 domain, (2) characterization of the ts defect in viral RNA synthesis, (3) identification of cellular and viral proteins which bind the 5NTR in the region of the ts mutations, and (4) construction and evaluation of additional mutants with more robust ts phenotypes. The long-term goals of these studies are an improved understanding of the cap-independent initiation of translation by picornaviral 5NTRs and the development of new attenuated hepatitis A vaccine candidates.
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