The long-term goals of this research are to understand how the herpes simplex virus major DNA-binding proteins, ICP8, moves into and within the cell nucleus and interacts with specific structures and molecules to promote viral DNA replication. This proposal focuses on the mapping of ICP8 domains for DNA- binding, nuclear localization and nuclear retention; the characterization of interactions of ICP8 with the sites of the host cell DNA synthesis; the role of ICP8 in defining the intranuclear location of parental and progeny DNA: and the identification of viral gene products needed to form replication compartments in transfected cells. The DNA-binding region on ICP8 will be identified by sequencing of ts mutant genes that encode ICP8 molecules that localize normally but are thermolabile for DNA binding. Linker insertions and oligonucleotide-directed mutations will also be constructed to identify portions of ICP8 needed for DNA-binding. To map nuclear localization signals, protein fusions will be made to identify the minimal portion of ICP8 needed to target pyruvate kinase to the cell nucleus. Putative nuclear retention signals will be mapped by mutagenesis of a truncated form of ICP8 that appears to be able to diffuse into and accumulate in the nucleus. To probe the function of cell proteins at the prereplicative sites, monoclonal antibodies will be prepared to the ICP8-cell protein complexes. The 3-dimensional distribution of ICP8 in the prereplicative sites will be defined by analysis of the fluorescent image at different focal depths. The role of ICP8 in inhibition of host cell DNA synthesis will be defined in infected and transfected cells. In situ hybridization using biotinylated probes will be used to localize viral DNA and examine the role of ICP8 in defining the nuclear distribution of viral DNA. Using transient expression assays, the minimal number of viral genes needed for assembly of replication compartments will be determined. This will identify any gene products specifically involved in assembly of these structures. These studies may provide information about the organization of the cell nucleus for viral and cellular DNA synthesis. The studies may provide a molecular explanation of how a virus takes over the cell nucleus for replication of its own genome. Thus, they may provide some insight into mechanisms controlling cell DNA replication.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA026345-12
Application #
3482056
Study Section
Virology Study Section (VR)
Project Start
1979-07-01
Project End
1992-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Uprichard, Susan L; Knipe, David M (2003) Conformational changes in the herpes simplex virus ICP8 DNA-binding protein coincident with assembly in viral replication structures. J Virol 77:7467-76
Taylor, Travis J; McNamee, Elizabeth E; Day, Cheryl et al. (2003) Herpes simplex virus replication compartments can form by coalescence of smaller compartments. Virology 309:232-47
Taylor, Travis J; Knipe, David M (2003) C-terminal region of herpes simplex virus ICP8 protein needed for intranuclear localization. Virology 309:219-31
Zhou, Changhong; Knipe, David M (2002) Association of herpes simplex virus type 1 ICP8 and ICP27 proteins with cellular RNA polymerase II holoenzyme. J Virol 76:5893-904
Brockman, Mark A; Knipe, David M (2002) Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity. J Virol 76:3678-87
Da Costa, X J; Morrison, L A; Knipe, D M (2001) Comparison of different forms of herpes simplex replication-defective mutant viruses as vaccines in a mouse model of HSV-2 genital infection. Virology 288:256-63
McNamee, E E; Taylor, T J; Knipe, D M (2000) A dominant-negative herpesvirus protein inhibits intranuclear targeting of viral proteins: effects on DNA replication and late gene expression. J Virol 74:10122-31
Da Costa, X; Kramer, M F; Zhu, J et al. (2000) Construction, phenotypic analysis, and immunogenicity of a UL5/UL29 double deletion mutant of herpes simplex virus 2. J Virol 74:7963-71
de Bruyn Kops, A; Uprichard, S L; Chen, M et al. (1998) Comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions. Virology 252:162-78
Uprichard, S L; Knipe, D M (1997) Assembly of herpes simplex virus replication proteins at two distinct intranuclear sites. Virology 229:113-25

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