The overall objectives of this project are to designate human cytomegalovirus (CMV)-specific gene functions and to express DMV-specific antigens in prokaryotic and eukaryotic systems for future development of diagnostic antigens and the CMV subunit viral vaccine in order to prevent and control cytomegalovirus infection. The long term objectives will be achieved through the following specific approaches. (1) To continue to construct the detailed restriction enzymes cleavage map of L repeat, S repeat and L-S junction fragments of DMV genome, and to search for and characterize the possible putative """"""""a"""""""" and """"""""c"""""""" sequence in CMV genomes by nucleic acid hybridization and DNA sequencing techniques. The possible origin of DNA replication in putative """"""""c"""""""" sequence will be examined in yeast system and in human fibroblast for its autonomous replication sequence. (2) To map genes coded for the virus-induced DNA polymerase, ribonucleoside reductase, virus-specific structural polypeptides (including 94K, 150K and other glycopolypeptides, etc.) using various expressive vectors of E. coli (pDR540, pDR720, pVE-J001 and Lambdagt11 and of mammalian cell (e.g. pSVOH in COS-1 cell), and various available monoclonal antibodies generated in our laboratory. (3) To complete the classification and characerization of monoclonal antibodies generated against purified virus and infected cell lysate for gene cloning and the development of diagnostic reagent. (4) To express CMV specific non-glycosylated polypeptides in E. coli using expression vectors which carry strong tac and trp promoters (e.g. pDR540, pDR720 and pVEJ1001), and glycosylated (also non-glycosylated) polypeptides in yeast using expression vectors carrying a strong alcohol dehydrogenase promoter (pAAH-5 and pMA56). Finally, (5) the viral polypeptides which are responsible for the induction of a major humoral immune response will be determined by Western blotting, immunoprecipittion, and SDS-PAGE. The antigenic determinants will be studied further by proteolytic digestion and Western blot immunoreaction. The DNA fragments encoding these polypeptides will be engineered and cloned for studying the feasibility of developing the subunit viral vaccine.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012717-13
Application #
3125268
Study Section
Virology Study Section (VR)
Project Start
1978-06-01
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1991-06-30
Support Year
13
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Johnson, R A; Huong, S M; Huang, E S (2000) Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38. J Virol 74:1158-67
Yurochko, A D; Huang, E S (1999) Human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression. J Immunol 162:4806-16
Yurochko, A D; Huong, S M; Huang, E S (1999) Identification of human cytomegalovirus target sequences in the human immunodeficiency virus long terminal repeat. Potential role of IE2-86 binding to sequences between -120 and -20 in promoter transactivation. J Hum Virol 2:81-90
Johnson, R A; Huong, S M; Huang, E S (1999) Inhibitory effect of 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H - imidazole on HCMV DNA replication and permissive infection. Antiviral Res 41:101-11
Johnson, R A; Yurochko, A D; Poma, E E et al. (1999) Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. J Gen Virol 80 ( Pt 5):1293-303
Yurochko, A D; Hwang, E S; Rasmussen, L et al. (1997) The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection. J Virol 71:5051-9
Yurochko, A D; Mayo, M W; Poma, E E et al. (1997) Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kappaB promoters. J Virol 71:4638-48
Poma, E E; Kowalik, T F; Zhu, L et al. (1996) The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol 70:7867-77
Yurochko, A D; Kowalik, T F; Huong, S M et al. (1995) Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters. J Virol 69:5391-400
Wing, B A; Huang, E S (1995) Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99. J Virol 69:1521-31

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