Recombinant HCMV AD169 viruses were reconstituted from transfection of corresponding BAC-HCMV clones as previously described [34] . Viral stocks were prepared by ultra-centrifugation of infected culture supernatant through 20% D-sorbitol cushion and re-suspending pelleted virus in serum-free medium. The following BAC-HCMV clones were used in the present study, and were constructed using PCR-based linear recombination as previously reported [29] , unless indicated otherwise. pAD-GFP, which carried the GFP-tagged genome of the HCMV AD169 strain, was used to produce wild-type virus ADgfp [45] . pADgfpUL21a, which carried an N-terminally GFP-tagged version of pUL21a, was used to produce ADgfpUL21a virus [29] . pADsubUL21a, which carried a GalK/kanamycin dual mutagenic cassette in place of the UL21a coding sequence, was used to produce UL21a-deletion virus ADsubUL21a [29] . pADgfpU-L21a PR-AA , pADgfpUL21a PH-AA , pADpmUL21a PR-AA , or pADp-mUL21a PH-AA carried point mutation PR109-110AA or PH111-112AA in the GFP tagged or native UL21a gene, respectively. These recombinant BAC clones were used to produce corresponding point mutant viruses. pADpmUL21a PH-AA /subUL97 and pADpmUL21a PR-AA /subUL97 carried the GalK/kanamycin mutagenic cassette in place of UL97 on the background of pADpmUL21a PR-AA and pADpmUL21a PH-AA BAC clones. Similarly, pADpmUL21a PH-AA /subUL117, pADpmUL21a PR-AA / subUL117, and pADsubUL117 carried the GalK/kanamycin mutagenic cassette in place of UL117 on the background of pADpmUL21a PR-AA , pADpmUL21a PH-AA , and pAD-GFP BAC clones, respectively. All BACs were confirmed by restriction digestion, PCR, and sequencing. HCMV virus titers were determined in duplicate in HFFs by tissue culture infectious dose 50 (TCID 50 ) assay or plaque assay. Relative viral genome numbers were determined by real-time quantitative PCR (qPCR) as described previously [29] .