Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct 22;110(43):17510-5.
doi: 10.1073/pnas.1312235110. Epub 2013 Oct 7.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state

Boris Bogdanow  1 , Henry Weisbach, Jens von Einem, Sarah Straschewski, Sebastian Voigt, Michael Winkler, Christian Hagemeier, Lüder Wiebusch
Affiliations

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state

Boris Bogdanow et al. Proc Natl Acad Sci U S A. .

Abstract

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HCMV-pp150 is a cyclin A2–CDK substrate. (A) pp150 is composed of an N-terminal capsid-binding domain and a large C-terminal part that contains a prototypical cyclin A2-binding motif and a number of consensus CDK-phosphoacceptor sites. (B) Alignment of the RRLFG motif of pp150 with a selection of functional RXL motifs of CDK inhibitors and CDK substrates. Identical residues are highlighted in black and conserved residues in gray. (C) Coomassie-staining of His-purified, recombinant C-terminal fragments (amino acids 303–1049) of pp150 wild-type (His-pp150c-WT) and pp150-RRLFGAAAAA mutant (His-pp150c-RXLmut). (D and E) Immobilized His-pp150c proteins were incubated with HEK293 cell lysates containing ectopically expressed HA-cyclin A2 wild-type (CycA2-WT), HA-cyclin A2 hydrophobic patch mutant (CycA2-hpm), HA-cyclin B1 (CycB1), or GFP. The input lysates and the pulled down material were analyzed by immunoblotting (IB) for the presence of pp150 and HA-tagged cyclins. (F) Kinase assays. Cyclin A2- and cyclin B1-associated kinases were immunoprecipitated from HEK293 lysates and incubated with the indicated substrate proteins.
Fig. 2.
Fig. 2.
The RRLFG motif in pp150 is essential for the CDK-dependent block of IE gene expression in S/G2 and cyclin A2-overexpressing cells. Proliferating HEL fibroblasts (A) and lentiviral vector-transduced U373 cells (B) were infected with the indicated recombinant viruses (MOI = 5), all based on HCMV strain TB40/e. CycA2ΔD, transduction of a cyclin A2 destruction box mutant; CycA2ΔD(R211A), transduction of a CycA2ΔD mutant deficient in CDK binding. At 5 h postinfection, cells were analyzed for DNA and IE1/2 protein content by flow cytometry. Four subpopulations were defined: IE-positive G0/G1 cells (upper left quadrant), IE-positive S/G2 cells (upper right quadrant), IE-negative G0/G1 cells (lower left quadrant), and IE-negative S/G2 cells (lower right quadrant). The percentages of each subpopulation are indicated.
Fig. 3.
Fig. 3.
The pp150-mediated cell cycle dependency is a distinctive feature of HCMV. (A) Schematic representation of pp150 and its mammalian CMV homologs of chimpanzee (C), rhesus (Rh), simian (S), guinea pig (GP), rat (R), and murine (M) origin. Based on the degree of conservation and a disorder tendency score (see upper panel), pp150 can be divided into two parts: a conserved, structured capsid-binding domain and a variable, intrinsically disordered C-terminal region. Depicted are the positions of sequence elements matching the consensus of CDK-phosphoacceptor sites or cyclin A2-binding motifs (φ: bulky hydrophobic residue). The total numbers of amino acids (aa) are indicated on the right. (B) Sequence alignment of the RRLFG-containing region of pp150 with the corresponding region in CCMV-pUL32. (C) Proliferating HEL fibroblasts were infected with HCMV or CCMV and analyzed for DNA content and IE1/2 gene expression as described in the legend of Fig. 2.
Fig. 4.
Fig. 4.
Mutation of the pp150-RRLFG motif overcomes the CDK-dependent block of IE gene expression in undifferentiated cells. NT2 cells were infected with the indicated HCMV recombinants. Where indicated cells were differentiated by retinoic acid (RA) treatment before infection. Trichostatin A (TSA), CDK inhibitors SU9516, CVT313 and Roscovitine (Rosco), or solvent (DMSO) were added together with the virus. At 24 h postinfection, cells were analyzed for Oct3/4 and IE1/2 gene expression by flow cytometry. Differentiated (Oct3/4-negative) cells were excluded from analysis except in the case of RA treatment. The histogram shows the percentage of IE1/2-positive undifferentiated cells. Data are presented as means and SDs of biological triplicates.
Fig. 5.
Fig. 5.
pp150 is a cis-acting regulator of IE gene expression. (A and B) Cyclin A2-overexpressing and U373 control cells (as described in the Fig. 2 legend) were synchronized in G1 by contact inhibition and then infected with equal amounts of (A) RV2042 and RV3301 or (B) pp150-GFP and pp150-RFP expressing HCMV-TB40 derivatives. RV2042 encodes an IE2-FP611 fusion protein, RV3301, the pp150-RXL-mutant as described in the Fig. 2 legend. (A) At 4 h postinfection, IE2, cyclin A2, and GAPDH protein level were analyzed by immunoblotting. (B) At 3 h postinfection, cells were analyzed by confocal immunofluorescence microscopy for pp150-GFP (green foci) and pp150-RFP (red foci) localization. Nuclei were counterstained by DAPI. Representative images are shown (B, Upper). Based on at least 50 randomly selected cells per sample, the average percentages of nuclear or cytoplasmic-localized red, green and yellow (merged) foci were quantified (B, Lower). Nuclear localization of pp150 was defined by its fluorescence overlap with DAPI. (C) Schematic representation of pp150 as a cyclinA2–CDK-dependent, cis-acting molecular switch controlling the onset of IE gene expression from the parental viral genome.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kalejta RF (2013) Pre-immediate early tegument protein functionsed. Cytomegaloviruses: From Molecular Pathogenesis to Intervention, ed Reddehase MJ, (Caister Academic, Norfolk, UK), Vol 1, Chap 9, pp141–151.
    1. Gönczöl E, Andrews PW, Plotkin SA. Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells. Science. 1984;224(4645):159–161. - PubMed
    1. Lukashchuk V, McFarlane S, Everett RD, Preston CM. Human cytomegalovirus protein pp71 displaces the chromatin-associated factor ATRX from nuclear domain 10 at early stages of infection. J Virol. 2008;82(24):12543–12554. - PMC - PubMed
    1. Saffert RT, Kalejta RF. Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression. J Virol. 2006;80(8):3863–3871. - PMC - PubMed
    1. Saffert RT, Kalejta RF. Human cytomegalovirus gene expression is silenced by Daxx-mediated intrinsic immune defense in model latent infections established in vitro. J Virol. 2007;81(17):9109–9120. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources