Deficiencies in Cellular Processes Modulated by the Retinoblastoma Protein Do Not Account for Reduced Human Cytomegalovirus Replication in Its Absence

Abstract

Despite encoding multiple viral proteins that modulate the retinoblastoma (Rb) protein in a manner classically defined as inactivation, human cytomegalovirus (HCMV) requires the presence of the Rb protein to replicate efficiently. In uninfected cells, Rb controls numerous pathways that the virus also commandeers during infection. These include cell cycle progression, senescence, mitochondrial biogenesis, apoptosis, and glutaminolysis. We investigated whether a potential inability of HCMV to regulate these Rb-controlled pathways in the absence of the Rb protein was the reason for reduced viral productive replication in Rb knockdown cells. We found that HCMV was equally able to modulate these pathways in the parental Rb-expressing and Rb-depleted cells. Our results suggest that Rb may be required to enhance a specific viral process during HCMV productive replication. R etinoblastoma (Rb) protein function is modified by multiple viruses (1-3). Through transcriptional repression of the E2F-responsive genes required for DNA replication, hypophosphorylated (active) Rb impedes cell cycle transit through G 1 and into S phase (4). Rb can also induce the formation of heterochromatin at E2F responsive genes, leading to permanent transcriptional silencing and replicative senescence (5, 6), providing a tumor suppressive function. As the role of Rb as a mediator of senescence and restrictor of cell cycle progression has long been acknowledged, the prevailing model in the field of DNA virology has associated viral targeting of Rb with maintaining a cell cycle state conducive to viral replication (7). Specifically, it was proposed that viruses alter the function of Rb to provide an S-phase-like environment where the enzymes and small molecule precursors necessary for DNA synthesis would be readily available for viral DNA replication. Indeed, the ability of the E7 protein of the high-risk human papillomavirus strain 16 to bind Rb is necessary for viral DNA replication (8).However, we recently reported that transient and stable Rb knockdown reduces the efficiency of human cytomegalovirus (HCMV) DNA synthesis and productive replication (9). This result was unexpected as HCMV encodes at least four viral proteins reported to modify several biological functions of Rb (2). Therefore, the relationship between viruses and Rb appears more complicated than the current paradigm allows.In recent years Rb has been shown to affect many facets of mitochondrial function in addition to its critical role in controlling the cell cycle. These include mitochondrial biogenesis, apoptosis, and the utilization of glutamine for the tricarboxylic acid (TCA) cycle and the production of glutathione. In the absence of Rb, cells have lower ratios of mitochondrial to cellular DNA, and this has been ascribed to defects in mitochondrial biogenesis (10,11). Rb regulates apoptosis directly at the mitochondria by binding to Bax (12,13). Interestingly, it is a phosphorylated form of Rb that interacts with Bax, and loss of this form can trigger apoptosis (12...

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins

“…The artificial depletion of p107 did not inhibit HCMV productive replication (60). Unlike Rb and p130 (6,8), p107 is not a strong tumor suppressor (73), and therefore it may affect processes less important for controlling both oncogenesis and viral infection.…”

“…1) and inactivated ( Fig. 4) by UL97, is not required for HCMV infection (60). Because p130 had yet to be tested, we monitored HCMV infectious progeny formation in cells in which p130 expression was stably reduced by shRNA-mediated interference.…”

“…5). We have not tested longer time points but predict the observed decrease in replication would persist because it does in Rbknockdown cells (60). Likewise, because a clinical strain virus also shows decreased replication in Rb-knockdown cells (59), we suspect one would also show defects in p130-knockdown cells, but we have not performed this experiment.…”

Human cytomegalovirus lytic infection inhibits replication-dependent histone synthesis and requires stem loop binding protein function

“…The current dogma proposes that DNA viruses including HCMV drive quiescent cells into the S phase, in part by inactivating the retinoblastoma (Rb) tumor suppressor, in order to accumulate the macromolecules and enzymes required for viral DNA synthesis. However, the Rb protein plays an unidentified positive role during HCMV infection ( 58 , 59 ), and the accumulation of deoxyribonucleotides for DNA synthesis does not appear to explain why HCMV induces cell cycle progression ( 60 ). Our revelation that SLBP localization and function is modulated during HCMV productive replication presents an opportunity to revisit the role of cell cycle modulation during DNA virus infections.…”