As two HCMV proteins, pUL97 and pUL21a, are capable of regulating the APC, we hypothesized that one of these two proteins acted to compensate for the loss of the other during infection. Consistent with this hypothesis, HCMV appeared to retain the ability, at least to some extent, to regulate the APC even when pUL21a or pUL97 is absent ( Figure 4E , and data not shown) [24] . To test this hypothesis more directly, we created recombinant HCMV viruses ADpmUL21a PH-AA /subUL97 and ADpmU-L21a PR-AA /subUL97. The two viruses were derived from AD-pmUL21a PH-AA and ADpmUL21a PR-AA , respectively, and both contained an additional deletion in UL97. Both recombinant viruses grew slower than wild-type virus due to lack of the multifunctional pUL97 protein ( Figure 6C ). However, reconstitution of ADpmUL21a PR-AA /subUL97 that lacked pUL21a APCbinding activity following BAC transfection was markedly slower than that of ADpmUL21a PH-AA /subUL97 ( Figure 6B ). At day 25 post transfection, while cells transfected with the BAC clone of ADpmUL21a PH-AA /subUL97 showed nearly 100% of CPE indicated by virus-driven GFP expression, GFP-positive foci in cells transfected with the BAC clone of ADpmUL21a PR-AA /subUL97 were distinctly smaller. Furthermore, multi-step growth curve analysis showed that titers of ADpmUL21a PR-AA /subUL97 were 13-and 14-fold lower than that of ADpmUL21a PH-AA /subUL97 at 14 and 21 days post infection (dpi), respectively ( Figure 6C ). As a control to show that this phenotype was not due to general viral attenuation resulting from the UL97 deletion, we also constructed double mutant viruses ADpmUL21a PH-AA /subUL117 and AD-pmUL21a PR-AA /subUL117. These two viruses were derived similarly from ADpmUL21a PH-AA and ADpmUL21a PR-AA , but also contained a deletion in viral gene UL117. We chose UL117 as the control because its mutation attenuated virus growth but not viral early or early-late gene expression so UL97 expression was unlikely affected [34] . BAC transfection reconstituted both mutant viruses at similar efficiency and produced viruses with similar titers (data not shown). Multi-step growth analysis demonstrated that ADpmUL21a PH-AA /subUL117 and ADpmUL21a PR-AA /subUL117 Figure 6 . Abrogation of both pUL21a APC regulatory activity and pUL97 results in a more severe attenuation in HCMV growth than pUL97 deletion alone. (A) Abrogation of pUL21a-APC binding alone is not sufficient to alter HCMV replication. MRC-5 cells in serum-containing (cycling condition) or serum-free (G0 condition) media were infected with ADgfp, ADsubUL21a, ADpmUL21a PH-AA , or ADpmUL21a PR-AA at an MOI of 0.01. Production of cell-free virus at indicated times was determined by plaque assay. (B) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced the efficiency of HCMV reconstitution as compared to abrogation of UL97 alone. To reconstitute ADpmUL21a PR-AA /subUL97 and pADpmUL21a PH-AA /subUL97 viruses, MRC-5 fibroblasts were transfected with their corresponding BAC clones. For each recombinant virus, three independent clones were tested. Shown are representative images of virus spread indicated by virus-driven GFP expression at indicated days post transfection of two of the three clones. Images were taken under a Leica fluorescent microscope. (C) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced HCMV replication as compared to abrogation of UL97 alone. MRC-5 cells were infected with indicated recombinant viruses at an input genome number equivalent to that of 0.03 infectious units of wild type virus/cell. Production of cell-free virion DNA at indicated times was determined by qPCR analysis and normalized to input levels of ADpmUL21a PH-AA , which was set to 1. (D) Multi-step growth analysis of double mutant viruses that carried the UL117 deletion and point mutation in the UL21a-APC binding site. Cells were infected with indicated recombinant viruses and analyzed as described in panel C. The input value of ADgfp was set to 1. doi:10.1371/journal.ppat.1002789.g006 replicated at similar kinetics ( Figure 6D ). At 14 dpi, the titer of ADpmUL21a PR-AA /subUL117 was slightly lower (e.g. 1.5-fold) than that of ADpmUL21a PH-AA /subUL117. However, growth of mutant virus carrying only the UL117 deletion tracked with ADpmUL21a PR-AA /subUL117, suggesting that the difference between the PH and PR mutants at 14 dpi, if any, is minimal. Together, our data provide evidence that disruptions of both pUL97 and the APC regulatory activity of pUL21a are synthetically lethal to HCMV replication. This is consistent with a working model that these two functions enable HCMV to cope with APC activity to promote virus replication (Figure 7) .