Infections with Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) constitute a major health problem in the United States. This proposal aims to purify and characterize proteins present in HSV infected cells which are involved in the replication and expression of the viral DNA. We will continue our work on the major HSV DNA binding (ICP8). Affinity constants and stoichiometries of binding will be obtained for the interaction of ICP8 with single and double-stranded nucleic acids. We will also determine the rate of exchange of this protein between nucleic acids. The various functional domains of ICP8 will be mapped by means of specific modification of individual amino acid residues and by studies on the properties of ICP8 molecules containing temperature sensitive lesions in various locations. The effect of the presence of ICP8 on homologous (HSV) and heterologous (mammalian, yeast and procaryotic) DNA poloymerases will be determined. A DNA primase activity which has been identified in HSV-infected cells will be isolated and characterized. The purified primase activity will be used in an attempt to reconstruct the HSV replication apparatus. Z-DNA binding proteins and Z-DNA suequences in the HSV system will be identified and isolated. The Z-DNA binding proteins will be tested for binding specifi- city using cloned HSV restriction enzyme fragments. Z-DNA sequences present in HSV DNA will be tested for enhancer activity using a chloram- phenicol transacetylase (CAT) assay. Methodology used will include: fluorescence spectroscopy, filter binding, molecular cloning, DNA agarose/ cellulose chromatography and Z-DNA immunoaffinity assays.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI022468-04
Application #
3133566
Study Section
Experimental Virology Study Section (EVR)
Project Start
1985-09-30
Project End
1993-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20852
Dudas, K C; Scouten, S K; Ruyechan, W T (2001) Conformational change in the herpes simplex single-strand binding protein induced by DNA. Biochem Biophys Res Commun 288:184-90
Dudas, K C; Ruyechan, W T (1998) Identification of a region of the herpes simplex virus single-stranded DNA-binding protein involved in cooperative binding. J Virol 72:257-65
Shelton, L S; Albright, A G; Ruyechan, W T et al. (1994) Retention of the herpes simplex virus type 1 (HSV-1) UL37 protein on single-stranded DNA columns requires the HSV-1 ICP8 protein. J Virol 68:521-5
Ruyechan, W T; Olson, J W (1992) Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA. J Virol 66:6273-9
Hay, J; Ruyechan, W T (1992) Regulation of herpes simplex virus type 1 gene expression. Curr Top Microbiol Immunol 179:1-14
Gupte, S S; Olson, J W; Ruyechan, W T (1991) The major herpes simplex virus type-1 DNA-binding protein is a zinc metalloprotein. J Biol Chem 266:11413-6
Haffey, M L; Stevens, J T; Terry, B J et al. (1988) Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. J Virol 62:4493-8
Ruyechan, W T (1988) N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein. J Virol 62:810-7
Holmes, A M; Wietstock, S M; Ruyechan, W T (1988) Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells. J Virol 62:1038-45
Ruyechan, W T; Chytil, A; Fisher, C M (1986) In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein. J Virol 59:31-6