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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032599-07
Application #
2886748
Study Section
Virology Study Section (VR)
Program Officer
Johnson, Leslye D
Project Start
1993-01-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
7
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Yi, MinKyung; Lemon, Stanley M (2002) Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. J Virol 76:1171-80
Beard, M R; Cohen, L; Lemon, S M et al. (2001) Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction. J Virol 75:1414-26
Blank, C A; Anderson, D A; Beard, M et al. (2000) Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes. J Virol 74:6476-84
Lerat, H; Shimizu, Y K; Lemon, S M (2000) Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells. J Virol 74:7024-31
Gosert, R; Chang, K H; Rijnbrand, R et al. (2000) Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo. Mol Cell Biol 20:1583-95
Yi, M; Schultz, D E; Lemon, S M (2000) 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. J Virol 74:6459-68
Honda, M; Rijnbrand, R; Abell, G et al. (1999) Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site. J Virol 73:4941-51
Honda, M; Beard, M R; Ping, L H et al. (1999) A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J Virol 73:1165-74
Martin, A; Benichou, D; Chao, S F et al. (1999) Maturation of the hepatitis A virus capsid protein VP1 is not dependent on processing by the 3Cpro proteinase. J Virol 73:6220-7
Hardin, C C; Sneeden, J L; Lemon, S M et al. (1999) Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of hepatitis A virus. Nucleic Acids Res 27:665-73

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