To define the precise sequence of the APC binding site, we made gfpUL21a mutants in which each of five conserved residue clusters within its carboxyl terminus were individually substituted with alanine residues (Figure 2A ). As a control, we also made alanine substitutions for the non-conserved proline-histidine pair at residues 111-112 (PH111-112AA) ( Figure 2A ). All mutants were stable and were efficiently pulled down by the GFP antibody ( Figure 2D , and data not shown). Among them, only the PR109-110AA mutant lost the ability to bind to the APC. Substitutions of the adjoining non-conserved residues (PH111-112AA) had no effect on APC binding. To validate the result in the context of infection, we constructed recombinant HCMV viruses expressing GFP-tagged or native forms of PR109-110AA or PH111-112AA pUL21a variants (ADgfpUL21a PR-AA , ADgfpUL21a PH-AA , AD-pmUL21a PR-AA , and ADpmUL21a PH-AA ). During infection, a reciprocal interaction between gfpUL21a PH-AA and APC3 could be detected while gfpUL21a PR-AA and APC3 did not interact ( Figure 2E ). Furthermore, untagged pUL21a PH-AA , but not pUL21a PR-AA , was co-immunoprecipitated with APC3 when stabilized by MG132 ( Figure S2 ). Together, these results indicate that the carboxyl terminus of pUL21a contains the APC binding domain and the residues PR 109-110 are critical for this binding.