Cell Cycle Modulation


Part of our laboratory studies how the HCMV pp71 and UL97 proteins modulate the Rb-E2F pathway, and how this may contribute to HCMV replication and pathogenesis.


Our most recent cell cycle paper:

VanDeusen, H.R., and Kalejta, R.F. (2015) Deficiencies in cellular processes modulated by the retinoblastoma protein do not account for reduced human cytomegalovirus replication in its absence. J. Virol. 89, 11965-74 PMCID: PMC4645314 pdf >publications

A classic cell cycle paper from our lab:

Hume, A.J., Finkel, J.S., Kamil, J.P., Coen, D.M., Culbertson, M.R., and Kalejta, R.F.  (2008)  Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function.  Science, 320, 797-799.  PMCID: none pdf   >publications

One of our cell cycle review articles:

Dziurzynski, K., Cjang, S.M., Heimberger, A.B., Kalejta, R.F., McGregor Dallas, S.R., Smit, M., Soroceanu, L., Cobbs, C.S., and the HCMV and Gliomas Symposium.  (2012) Consensus on the role of human cytomegalovirus in glioblastoma. Neuro Oncol. 14, 246-255. PMCID: PMC3280809 pdf >publications

 

 

The mammalian cell cycle is composed of four phases, G1, S, G2, and M.  Extracellular growth signals stimulate progression through the first gap phase G1, and into S, where genomic DNA is replicated.  After S and G2, nuclear division and cytokinesis in mitosis (M) produces two daughter cells.  If insufficient growth signals are detected during G1 phase, cells can exit the cell cycle and enter a quiescent or resting state termed G0.  Cell cycle transit is controlled in both G1 and G2 phases by a family of cyclin-dependent kinases (CDKs) whose activity depends upon their association with the cyclin proteins.  Cyclin synthesis and degradation are tightly regulated which, along with the action of the cyclin-dependent kinase inhibitors (CKIs) contributes to the control of cell cycle progression. To drive cell cycle progression, different cyclin/CDK combinations phosphorylate their substrates, which include the retinoblastoma family of tumor suppressors, Rb, p107 and p130. 

The Rb proteins are inhibitors of the E2F family of transcription factors.  When bound by Rb proteins, E2Fs repress transcription because Rb recruits histone deacetylases to E2F-responsive promoters.  Because the protein products of many E2F-responsive genes are required for cell cycle progression out of quiescence (G0), through G1 and into the S phase, Rb-E2F complexes arrest cell cycle progression in G0/G1.  Phosphorylation of Rb by the CDKs disrupts HDAC-Rb-E2F complexes, relieving Rb-imposed repression of E2F-activated genes. The Rb-E2F pathway represents the major controlling force that regulates progression out of G0, through G1, and into the S phase.

E2F-repsonsive genes have historically been grouped into three general categories: cell cycle proteins (e.g. cyclin E), DNA replication enzymes (e.g. polymerases and helicases), and nucleotide biosynthetic enzymes (NBEs).  Thus, the proteins encoded by E2F-responsive genes drive cells through the G1 phase, generate the needed macromolecules for DNA synthesis, and polymerize DNA.

As obligate intracellular parasites, viruses expertly modify cellular processes to facilitate their life cycles, and have evolved efficient ways to inactivate Rb to create an advantageous cellular environment for viral replication. Mechanisms through which viruses inactivate Rb are: direct binding to Rb to disrupt Rb-E2F complexes, induction of Rb degradation, constitutive activation of cellular CDKs by virally encoded cyclins, and direct phosphorylation by virally-encoded protein kinases. 

Infection of quiescent human fibroblasts (HFs) with HCMV initiates a lytic infection and an abortive mitogenic response that leads to cell cycle stimulation but not to the synthesis of the host cell’s genome.  Cells are stimulated to exit G0 and traverse G1, but eventually arrest at the G1/S border.  This cell cycle position is presumably favorable for viral DNA replication because all of the building blocks required for DNA synthesis are present, but not utilized for the synthesis of the host cell’s genome.  The Rb-E2F pathway that controls progression out of G0, through G1, and into S phase is modulated during HCMV infection.

Hypophosphorylated Rb is not observed in HCMV infected cells, but the hyperphosphorylated form of Rb accumulates.  The lack of hypophosphorylated Rb results from both degradation and phosphorylation.  HCMV encodes four proteins (pp71, UL97, IE1, and IE2) that stimulate cell cycle progression.  pp71 degrades Rb, p107 and p130.  UL97 phosphorylates Rb, p107 and p130.  IE1 inactivates p107 by an unknown mechanism, and IE2 directly activates E2F-respopnsive gene expression.

pp71 is a tegument protein required for efficient replication at low multiplicities to activate expression of the viral IE genes by degrading the cellular Daxx protein (that silences IE gene expression) in a proteasome-dependent, ubiquitin-independent manner.  Interestingly, pp71-mediated Daxx degradation occurs only at the beginning of lytic infections, indicating that the ability of pp71 to mediate protein degradation may be attenuated as HCMV infection progresses. pp71 also stimulates cell cycle progression by inducing the proteasome-dependent, ubiquitin-independent degradation of the hypophosphorylated forms of the Rb proteins.  pp71 contains a near consensus Rb-binding motif (LxCxD) and a mutant (C219G) that contains a single cysteine to glycine change in that motif fails to degrade Rb and fails to induce DNA synthesis in quiescent cells.  pp71 is responsible for the degradation of the hypophosphorylated form of the Rb protein that is observed within the first four hours after HCMV infection of fibroblasts.  Interestingly, a virus expressing only the C219G mutant of pp71 grows with wild type kinetics, likely because HCMV encodes multiple redundant mechanisms to target the Rb-E2F pathway. 

UL97 is a serine-threonine kinase that augments, but is not absolutely required for HCMV lytic replication in fibroblasts in vitro. Deletion of the UL97 gene or inhibition of UL97 kinase activity results in a decrease in viral DNA replication, defects in virion assembly and egress, and the production of 10- to 1000-fold fewer infectious viral particles.  UL97 is also a key protagonist for the small arsenal of drugs available to treat HCMV infections.  UL97 is required to phosphorylate and thus activate the gancyclovir family of antiherpesvirus drugs, and UL97 itself is the target of Maribavir, a compound currently under development for the treatment of HCMV infections.  UL97 is responsible for the Rb phosphorylation that is observed in HCMV-infected cells. UL97-null viruses fail to induce Rb phosphorylation, indicating the UL97 is necessary for Rb phosphorylation during HCMV infection.  By itself, UL97 is sufficient to phosphorylate Rb both in vivo and in vitro.  Kinase activity was required for Rb phosphorylation, and phosphorylation events were observed on residues known to inactivate the cell cycle inhibitory and tumor suppressor functions of Rb.  Not surprisingly, UL97 stimulates cell cycle progression.  Interestingly, it displays bona fide CDK activity, but is not subject to control mechanisms that restrict cellular CDKs. Phosphorylation by a v-CDK is a newly-discovered means by which viruses inactivate Rb.  Inactivation of the Rb proteins must be a critical function of UL97 during HCMV infection because the papillomavirus wild type E7 protein that induces the degradation of all three Rb family members could partially rescue the growth of a UL97-null virus in quiescent (serum-starved) cells where UL97-null viruses show a 1000-fold growth defect.  However, a mutant E7 with a disrupted LxCxE motif that is unable to bind Rb could not rescue the growth of the UL97-null virus. 

Four putative Rb binding motifs are found in UL97, three LxCxE motifs and one hydrophobic patch.  The first LxCxE motif (L1) is required for full Rb phosphorylation and inactivation.  None of the other motifs are required.  The UL97-L1 mutant efficiently disrupts Rb-E2F complexes yet fails to reverse the transcriptional repression instituted by Rb.  This defect can be complemented by the viral IE1 protein.

HCMV IE1 is required for replication at low multiplicities of infection, and stimulates cell cycle progression, but only in p53-null or p21-null cells.  IE1, through its first 85 amino acids, interacts with the Rb family member p107, but not with Rb.  Although IE1 may interfere with some aspects of the Rb-E2F pathway, IE1 does not appear to be required for the induction of E2F-responsive genes during HCMV infection.

IE2 is absolutely required for lytic infection.  It binds Rb and the cyclin E promoter in vitro, activates E2F-responsive gene expression, and stimulates cell cycle progression.  Because an IE2 mutant deficient for transactivation but retaining the putative Rb-binding region fails to stimulate the cell cycle, it is likely that IE2 induces E2F genes independently of Rb.  However, any direct role for IE2 in the induction of E2F responsive genes during HCMV infection remains unestablished. Because UL97 is an early gene product, and because IE2 activates early gene expression, it seems that IE2 may induce E2F genes in two ways: by directly activating E2F-responsive promoters, and by inducing the expression of UL97 which itself induces E2F-responsive genes by phosphorylating Rb, p107, and p130.

From the relevant data, a model for how HCMV infection modulates progression out of G0 and towards the G1/S border in fibroblasts can be assembled.  Tegument-delivered pp71 binds to and degrades the hypophosphorylated forms of the Rb proteins.  pp71 also degrades Daxx to facilitate the expression of viral immediate early proteins.  One of these proteins, IE2, stimulates E2F-mediated gene expression perhaps by directly activating E2F-responsive genes at the level of transcription through a process independent of Rb, and by activating the expression of UL97, a viral early gene.  UL97 then phosphorylates Rb and activates E2F-responsive genes.  IE1 may help sustain E2F-responsive gene expression by inactivating p107.   

Despite encoding multiple viral proteins that modulate Rb in a manner classically defined as inactivation, HCMV requires the presence of the Rb protein to replicate efficiently.  Because HCMV is equally able to modulate cellular Rb-controlled pathways in wild type and Rb-depleted cells, it is possible Rb may promote a viral process during HCMV lytic infection.