Hepatitis A virus (HAV) is a unique picornavirus which causes acute hepatitis in man. We are interested in studying the structure and function of the 5' nontranslated region (NTR) of HAV RNA and its role in viral translation and viral replication in cell culture. We developed a model of the secondary structure of the NTR based on identification of covariant nucleotide substitutions, cleavage by double- and single-strand specific RNases, and thermodynamic predictions. We plan to refine this model by (1) phylogenetic analysis of additional HAV strains, (2) RNase and chemical nuclease studies of secondary structure in the less well defined 5' half of the NTR, and (3) Fe(II)-EDTA strand scission mapping of surface features and RNA folding patterns. Our previous work indicates that the 5'NTR contains an internal ribosomal entry site (IRES) extending from 5' of base 354 to the initiator AUG. We plan to map the structural elements of this IRES by examining (1) in vitro translation of bicistonic constructs in which translation of the second cistron is controlled by 5'NTR elements, and (2) in vivo translation of mono- and bicistonic constructs in which expression of a reporter gene (CAT) is under control of 5'NTR elements in permissive cells. These experiments will utilize stably transfected BS-C-1 cells (BT7-6 cell line) which constitutively express the bacteriophage T7 RNA polymerase. We also demonstrated that mutations at several sites within the 5'NTR contribute to a highly host-cell specific enhancement of virus replication in BS-C-1 cells. One (and possibly two) of these sites is within the HAV IRES, suggesting that these mutations may enhance translation in vivo. We will test this hypothesis by examining translation of wt and mutant 5'NTR constructs in BT7-6 cells. Preliminary work indicates that a 39 kDa cytoplasmic protein of BS-C-1 cells (p39) binds specifically to two domains within the 5'NTR. These domains have limited sequence or structural relatedness, yet compete with each other for binding to p39.
We aim to (1) carry out a genetic analysis to determine the RNA structural requirements for binding by p39 in both domains, (2) determine whether p39 is part of the eIF-2/2B complex, and (3) assess whether 5'NTR mutations associated with enhanced growth in BS-C-1 cells alter affinity of RNA for p39. Because HAV translation follows binding of the 40S ribosomal subunit to the IRES, we postulate that HAV translation will continue and perhaps be enhanced in the presence of cytoplasmic expression of the poliovirus 2A protein, which should result in cleavage of the p220 component of eIF-4F and shut down translation of capped mRNAs. We will determine whether expression of poliovirus 2A in BT7-6 cells results in continued HAV translation, and influences yields of infectious HAV.
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