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Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1
Published on: August 24, 2015
Characterization of the single-stranded DNA-binding domain of the herpes simplex virus protein ICP8
1Department of Pathology, University of North Carolina, Chapel Hill.
Insights
Herpes simplex virus type 1 (HSV-1) DNA-binding protein ICP8 has three regions associated with single-stranded DNA (ssDNA) binding. Further analysis is needed to fully define the complete ssDNA-binding domain of ICP8.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Herpes simplex virus type 1 (HSV-1) DNA-binding protein ICP8 is essential for viral DNA replication.
- ICP8 exhibits multifunctional properties in vivo and a preference for binding single-stranded DNA (ssDNA) in vitro.
Purpose of the Study:
- To delineate the specific regions within ICP8 responsible for its ssDNA-binding activity.
- To identify the amino acid residues critical for DNA binding.
Main Methods:
- Cloning of ICP8 gene portions into a transcription vector.
- In vitro RNA synthesis and translation in rabbit reticulocyte lysates to produce ICP8 peptides (29, 35, and 30 kDa).
- Analysis of peptide ssDNA-binding activity using ssDNA-cellulose column chromatography.
Main Results:
- Three distinct regions of ICP8 (residues 332-564, 571-899, and 900-1196) demonstrated association with ssDNA-binding.
- Peptides corresponding to residues 332-564 and 571-899 showed significant ssDNA-binding activity.
- A larger peptide (95 kDa) encompassing these regions did not exhibit the same binding characteristics as intact ICP8, indicating the domain is not fully defined.
Conclusions:
- Specific regions of ICP8, including residues 332-564 and 571-899, are crucial for ssDNA binding.
- These identified regions also contain mutations found in ICP8 variants with impaired DNA-binding capabilities.
- The complete ssDNA-binding domain of ICP8 requires further investigation beyond these identified regions.
Abstract:
The DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is multifunctional in vivo and binds preferentially to single-stranded DNA (ssDNA) in vitro. To define the ssDNA-binding domain of ICP8, peptides were produced and analyzed. Portions of the ICP8 gene were cloned into the transcription vector pSP64, and RNA was synthesized in vitro. Translation of this RNA in rabbit reticulocyte lysates produced peptides of 29, 35 and 30 kDa, representing amino-acid residues 332-564, 571-899 and 900-1196, respectively, of intact ICP8 (128 kDa, 1196 amino acids). These peptides were analyzed by ssDNA-cellulose column chromatography. About 55% of the 29 kDa peptide bound to ssDNA-cellulose columns, and the majority which bound eluted with 1.0 M NaCl. About 5% of the 35 kDa peptide and 12% of the 30 kDa peptide bound and eluted with 0.3 M NaCl. Thus, three regions of ICP8 were associated with ssDNA-binding activity. The ssDNA-binding domain of ICP8 was not completely defined, however, because a 95 kDa peptide which included these regions did not bind to or elute from ssDNA-cellulose in the same way as intact ICP8. Amino-acid residues 332-564 and 571-899 not only were associated with ssDNA-binding activity but also contain the altered amino acids of four ICP8 molecules which are deficient in DNA binding.
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