Herpes Simplex Virus Type 1 ICP0 Protein Does Not Accumulate in the Nucleus of Primary Neurons in Culture

Abstract

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722–2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.