Identification of a motif in the C terminus of herpes simplex virus regulatory protein ICP4 that contributes to activation of transcription.

Expression of most viral genes during productive infection by herpes simplex virus is regulated by the viral protein ICP4 (also called IE175 or Vmw175). The N-terminal portion of ICP4 contains well-defined transactivation, DNA binding, and dimerization domains that contribute to promoter regulation. The C-terminal half of ICP4 contributes to the activity of ICP4, but the functional motifs have not been well mapped. To localize functional motifs in the C-terminal half of ICP4, we have compared the relative specific activities of ICP4 variants in transient-transfection assays. Deletion of the C-terminal 56 residues reduces the specific activity more than 10-fold. Mutational analysis identified three consecutive residues (1252 to 1254) that are conserved in ICP4 orthologs and are essential for full activity, especially in the context of ICP4 variants with a deletion in the N-terminal transactivation domain. Recombinant viruses that encode variants of ICP4 with mutations in the N-terminal transactivation domain and/or the extreme C terminus were constructed. The phenotypes of these recombinant viruses support the hypothesis that efficient promoter activation by ICP4 requires motifs at both the N and C termini. The data suggest that the C terminus of ICP4 functions not as an independent transactivation domain but as an enhancer of the ICP4 N-terminal transactivation domain. The data provide further support for the hypothesis that some ICP4 motifs required for promoter activation are not required for promoter repression and suggest that ICP4 utilizes different cellular factors for activation or repression of viral promoters.     

Mutational analysis of the att DNA-binding domain of phage Mu transposase.

The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.