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 wild-type 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.

Fig. 1.

UL97-null HCMVs that express wild-type or mutant forms of the E7 oncoprotein of HPV16. Schematic of the coding content of the UL95–UL105 region of the viruses used in this study. (A) Wild-type HCMV strain AD169 (WT); BamHI sites are labeled and an arrow representing the UL97 ORF is shown in black (top). (B) The same region of Δ97, a UL97 deletion virus is shown. A remnant 37-amino acid ORF, comprised of the first 23 amino acids of UL97 and an additional 14 amino acids attributed to a frameshift after the deletion, is shown in black; a gray box represents a remnant UL97 coding region not predicted to be translated due to a naturally occurring stop codon. (C) Δ97-E7, a UL97-null HCMV that encodes the E7 protein of HPV16 (E7, black arrow) in place of UL97. (D) Δ97-DLYC, a UL97-null HCMV that encodes a ΔDLYC mutant form of HPV16 E7 (DLYC, black arrow) in place of UL97.

Fig. 2.

Analysis of viral and cellular protein expression in quiescent cells. Western blot analyses of cellular and viral protein expression in cells infected with the viruses used in this study. (A) Serum-deprived human fibroblasts were infected at an MOI of 1 PFU/cell with WT HCMV (WT), UL97-null HCMV (Δ97), or UL97-null HCMV expressing either wild-type (Δ97-E7) or ΔDLYC mutant (Δ97-DLYC) forms of HPV16 E7. Lysates prepared at the indicated time points (hpi) were compared for the expression of the following viral gene products: the immediate early protein IE1–72 (IE1); UL57, an early protein; and pp28, a late protein. Expression levels of TS and PCNA were measured as examples of E2F-responsive cellular genes. Expression of UL97, E7, pRb were also monitored as controls, as was beta-actin, to indicate protein loading. Phosphospecific antibodies were used to monitor for the presence of pRb phosphorylated at Ser-807/811 (S807^P) and at Ser-780 (S780^P). (B) Serum-starved cells maintained in 0.1% FBS were either mock-infected (m) or infected with the same viruses and lysed at the indicated time points (hpi) following infection and analyzed for pRb expression and mobility by Western blot. Beta tubulin (tub) reactivity was determined as a loading control.

Fig. 3.

Comparison of mRNA levels for TS and UMPS. Analysis of mRNA levels for two genes involved in cellular DNA synthesis whose promoters bind E2F transcription factors. (A) RT-qPCR was used to compare mRNA levels at 48 hpi for TS and TATA-binding protein (TBP) in serum-deprived cells which were either mock-infected or infected with the indicated viruses. RNA from a set of cells that were serum stimulated in parallel and harvested at 24 h post-stimulation was used as a positive control for TS induction. TS mRNA levels are displayed as normalized values after correcting for differences in TBP expression. (B) mRNA levels at 48 hpi for UMPS were compared, as above. For both panels, the data represent the average measurement from three replicates per condition, with error bars representing standard deviations.

Fig. 4.

Complementation of a severe replication defect in quiescent cells of UL97-null HCMV by wild-type HPV E7. Replication kinetics experiments comparing viral replication efficiency in quiescent and cycling cells. (A) Serum-deprived cells, maintained in medium containing 0.1% FBS were infected with WT HCMV (WT), UL97-null HCMV (Δ97), or UL97-null HCMV expressing either wild-type (Δ97-E7) or ΔDLYC mutant (Δ97-DLYC) forms of HPV16 E7 at an MOI of 1 PFU/cell and compared for yield of infectious virus. (B) Asynchronously dividing cells, maintained in the presence of 5% FBS, were infected as above and compared for yield of infectious virus. For both panels, because counts from duplicate titrations did not vary by more than 2-fold for any data points, error bars are not shown.

Fig. 5.

Comparison of viral DNA synthesis in dividing versus non-dividing cells by Southern blot. Southern blot analysis of viral DNA synthesis in non-dividing, serum-deprived cells and asynchronously dividing cells maintained in 5% FBS. (A) Equal quantities of total DNA, prepared at the indicated time points from quiescent cells maintained in 0.1% FBS which were infected with WT HCMV (WT), UL97-null HCMV (Δ97), or UL97-null HCMV expressing either wild-type (Δ97-E7) or ΔDLYC mutant (Δ97-DLYC) forms of HPV16 E7, were analyzed by Southern blot using a probe against a viral gene (UL83). (B) Samples from a parallel experiment in asynchronously dividing cells maintained in 5% FBS were subjected to Southern blot analysis, as above. In both panels, a longer exposure is shown immediately beneath the normal exposure so that levels of DNA from input virus can be observed.