Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

Abstract

Several different families of DNA viruses encode proteins that inactivate the cellular retinoblastoma tumor suppressor protein (pRb), which normally functions to bind E2F transcription factors and restrict expression of genes necessary for cellular processes including DNA replication. Human cytomegalovirus (HCMV) UL97, a protein kinase functionally orthologous to cellular cyclin-dependent kinases, phosphorylates pRb on inactivating residues during HCMV infection. To assess if such phosphorylation is biologically relevant, we tested whether the human papillomavirus type 16 E7 protein, which inactivates pRb family proteins by direct binding and destabilization, could substitute for UL97 during HCMV infection. In the absence of UL97, expression of wild-type E7 protein, but not a mutant E7 unable to bind pRb family proteins, restored E2F-responsive cellular gene expression, late viral gene expression, and viral DNA synthesis to levels normally observed during wildtype virus infection of quiescent cells. UL97-null mutants exhibited more pronounced defects in virus production and DNA synthesis in quiescent cells as compared to serum-fed, cycling cells. E7 expression substantially enhanced infectious virus production in quiescent cells, but did not complement the defects observed during UL97-null virus infection of cycling cells. Thus, a primary role of UL97 is to inactivate pRb family proteins during infection of quiescent cells, and this inactivation likely abets virus replication by induction of cellular E2F-responsive genes. Our findings have implications for human cytomegalovirus disease and for drugs that target UL97.E2F ͉ cyclin-dependent kinase ͉ cell cycle ͉ antiviral drugs ͉ retinoblastoma protein

Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

“…Following this, PCR primers with sequences to facilitate homologous recombination into the viral genome (see supplemental Table S1 at our website, https://coen.med.harvard.edu) were used to amplify the D/N GFP-lamin A sequence containing the ISce-AphAI (KanaR) region from the plasmid pGFP-dhead-caax-KanS. The PCR product was gel purified and electroporated into GS1783 cells harboring either WT_E7 AD169rv (16) or WT AD169rv BAC. Kanamycin-resistant integrates were resolved by heat shock and L-(ϩ)-arabinose induction of I-SceI, and the resulting BACs were sequenced to confirm the introduced changes.…”

“…Kanamycin-resistant integrates were resolved by heat shock and L-(ϩ)-arabinose induction of I-SceI, and the resulting BACs were sequenced to confirm the introduced changes. For the WT HCMV construct (WT LMN ⌬H/C AD169rv), the mutated GFPlamin A sequences were cloned under the control of a duplicated UL97 promoter (described previously [16]) in the unique short (U S ) region of the HCMV genome within an intergenic locus between US9 and US10 by replacing the previously described human papillomavirus E7 gene cloned in that region (16). For the UL97 null (⌬97) HCMV construct (⌬97 LMN ␣-helical "rod" domains flanked by the head and tail domains, with a CaaX domain at the carboxyl terminus that is a site for isoprenylation.…”

“…Although UL97 has been implicated in several other steps during the virus replication cycle (10-16), in dividing cells the magnitude of the nuclear egress defect due to a UL97 null mutation or a UL97 inhibitor is very similar to the magnitude of the virus yield defect (10,17). In serum-starved, nondividing cells, part of the virus yield defect can be ascribed to a defect in viral DNA synthesis, stemming from UL97's role in inactivation of retinoblastoma protein (16)(17)(18). …”

“…In serum-starved, nondividing cells, part of the virus yield defect can be ascribed to a defect in viral DNA synthesis, stemming from UL97's role in inactivation of retinoblastoma protein (16)(17)(18). UL97 is necessary for the phosphorylation of a variety of viral and cellular proteins in infected cells (11,16,(18)(19)(20)(21)(22)(23)(24) and is sufficient for phosphorylation of some of these proteins in vitro (11,19,20,25,26). One such substrate is the nuclear lamina component lamin A/C.…”

“…Although UL97 has been implicated in several other steps during the virus replication cycle (10)(11)(12)(13)(14)(15)(16), in dividing cells the magnitude of the nuclear egress defect due to a UL97 null mutation or a UL97 inhibitor is very similar to the magnitude of the virus yield defect (10,17). In serum-starved, nondividing cells, part of the virus yield defect can be ascribed to a defect in viral DNA synthesis, stemming from UL97's role in inactivation of retinoblastoma protein (16)(17)(18). UL97 is necessary for the phosphorylation of a variety of viral and cellular proteins in infected cells (11,16,(18)(19)(20)(21)(22)(23)(24) and is sufficient for phosphorylation of some of these proteins in vitro (11,19,20,25,26).…”