Human Cytomegalovirus UL48 Deubiquitinase Primarily Targets Innermost Tegument Proteins pp150 and Itself To Regulate Their Stability and Protects Virions from Inclusion of Ubiquitin Conjugates.

Viral deubiquitinases (DUBs) regulate cellular innate immunity to benefit viral replication. In human cytomegalovirus (HCMV), the UL48-encoded DUB regulates innate immune responses, including NF-κB signaling. Although UL48 DUB is known to regulate its stability via auto-deubiquitination, its impact on other viral proteins is not well understood. In this study, we investigated the role of UL48 DUB in regulating the ubiquitination of viral proteins by comparing the levels of ubiquitinated viral peptides in cells infected with wild-type virus and DUB active-site mutants using mass spectrometry. We found that ubiquitinated peptides were increased in DUB mutant virus infection for 90% of viral proteins, with the innermost tegument proteins pp150 (encoded by UL32) and pUL48 itself being most significantly affected. The highly deubiquitinated lysine residues of pUL48 were mapped within its N-terminal DUB domain and the nuclear localization signal. Among them, the arginine substitution of lysine 2 (K2R) increased pUL48 stability and enhanced viral growth at low multiplicity of infection, indicating that K2 auto-deubiquitination has a role in regulating pUL48 stability. pUL48 also interacted with pp150 and increased pp150 expression by downregulating its ubiquitination. Furthermore, we found that, unlike the wild-type virus, mutant viruses expressing the UL48 protein with the DUB domain deleted or DUB active site mutated contain higher levels of ubiquitin conjugates, including the ubiquitinated forms of pp150, in their virions. Collectively, our results demonstrate that UL48 DUB mainly acts on the innermost tegument proteins pp150 and pUL48 itself during HCMV infection and may play a role in protecting virions from the inclusion of ubiquitin conjugates. IMPORTANCE Herpesviruses encode highly conserved tegument proteins that contain deubiquitinase (DUB) activity. Although the role of viral DUBs in the regulation of host innate immune responses has been established, their roles in the stability and function of viral proteins are not well understood. In this study, we performed a comparative analysis of the levels of ubiquitinated viral peptides between wild-type and DUB-inactive HCMV infections and demonstrated that the innermost tegument proteins pp150 and pUL48 (DUB itself) are major targets of viral DUB. We also show that ubiquitinated viral proteins are effectively incorporated into the virions of DUB mutant viruses but not the wild-type virus. Our study demonstrates that viral DUBs may play important roles in promoting the stability of viral proteins and inhibiting the inclusion of ubiquitin conjugates into virions.