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nggMSTiL

nggMSTiL

Fig. 2 Functional domains of the HCMV IE86 protein. a Abbreviations: AD activation domain, S serine domain, K lysine, T threonine, H histidine, P proline, Y tyrosine, Q glutamine, N nuclear localization signal, ZF putative zinc finger. b Amino acid sequence alignment of primate and nonprimate IE86 protein homologs. Identical amino acid residues are shaded in black and similar residues in gray. A star indicates a consensus sequence, and a dot a conserved sequence. A hyphen designates a gap in the sequence. MultAlin was used for the alignment of the IE86 residues for Towne strain (AAR31449), chimpanzee (NP612745), rhesus (AAB00488), African green monkey (AAB16881), mouse (AAA74505), and rat (AAB92266)

and 275 positively or negatively affects viral growth depending on the location of the residues (Barrasa et al. 2005) . Mutation of residues between 271 and 275 accelerates viral growth and mutation of residues between 258 and 264 or 266 and 269 delays viral growth (Table 1).

The IE86 protein has two nuclear localization signals (N) that can independently target the viral protein to the nucleus (Fig. 2a) (Pizzorno et al. 1991). In the nucleus, the viral protein affects viral promoters through two transcriptional activation domains, one amino and the other carboxyl (Fig. 2a) (Malone et al. 1990; Pizzorno et al. 1991; Yeung et al. 1993; Stenberg 1996). For transcriptional regulatory activity, the IE86 protein functions as a homodimer and dimerizes through the region broadly designated in Fig. 2a (Macias et al. 1996). Within this region, there is a putative zinc finger between amino acids 428 and 452, which may be part of a double zinc finger motif between amino acids 428 and 480 (CX5 CX11HX5HXDXCX13HXH) (Fig. 2a). Double zinc finger motifs are important for interactions with other viral or cellular proteins such as transcription factors that either activate or repress transcription (Bachy et al. 2002; Moreno et al. 2003).

Figure 2a summarizes the regions of posttranslational modification and the broadly mapped functional domains of the IE86 protein. Multiple amino acid deletions were made to determine the functional domains of the IE86 protein and these mutations are summarized in Table 1. Large deletions resulted in nonreplicating genomes and confirmed that the IE86 protein is essential for HCMV replication. Smaller deletions affected the efficiency of either early or late viral gene expression. Frequently more than one viral protein function was affected by these deletions. Mutations that affected DNA binding and negative autoregulation produced high levels of the IE86 protein, which also affected cell viability. Mutations that failed to interact with TBP or TFIIB affected activation of early viral promoters. Mutations that affected phosphorylation of the viral protein affected the rate of viral growth. Delayed viral growth was associated with reduced expression of viral tegument proteins pp65 and pp28 (Sanchez et al. 2002). Many of the mutations made it difficult to assign a particular function to a specific region of the IE86 protein. The region between amino acids 450 and 552 was defined as a core domain because the IE86 regulatory functions of negative autoregulation of the MIE promoter, early promoter transactivation, and cell cycle arrest were all affected by any deletion within this region (Asmar et al. 2004).

Figure 2b shows regions of conserved amino acids in the carboxyl end of the viral protein between primate and nonprimate CMV homologs of the IE86 protein. The carboxy terminus is more conserved than the amino terminus. There are conserved stretches of amino acids suggesting critical structural and functional domains within the protein. Petrik et al. made rationally designed amino acid substitutions in the core region based on sequence conservation (Petrik et al. 2006, 2007); these results are also summarized in Table 1. These mutations separated the transactivation domain from the cell cycle arrest domain and the autoregulation domain from the transactivation domain (Fig. 2a). However, mutations in the putative zinc finger motif affected all functions of the

Table 1 Effect of amino acid deletions or substitutions on the functions of the IE86 protein

Effect on function

Amino acids

Assay

Reference

Dimerization

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