Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

ABSTRACTHerpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with aUL97mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

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Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C, for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

Journal of Virology ◽

10.1128/jvi.02358-13 ◽

2013 ◽

Vol 88 (1) ◽

pp. 249-262 ◽

Cited By ~ 46

Author(s):

M. Sharma ◽

J. P. Kamil ◽

M. Coughlin ◽

N. I. Reim ◽

D. M. Coen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Cytomegalovirus ◽

Viral Protein ◽

Nuclear Lamina ◽

Nuclear Egress ◽

Kinase C ◽

Viral Protein Kinase ◽

Infected Cells

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Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1511140112 ◽

2015 ◽

Vol 112 (29) ◽

pp. 9010-9015 ◽

Cited By ~ 34

Author(s):

Kendra E. Leigh ◽

Mayuri Sharma ◽

My Sam Mansueto ◽

Andras Boeszoermenyi ◽

David J. Filman ◽

...

Keyword(s):

Nuclear Membrane ◽

Structural Information ◽

Point Mutations ◽

Nuclear Lamina ◽

Protein Protein Interaction ◽

Nuclear Egress ◽

Peptide Binding Site ◽

Infected Cells ◽

Nmr Methods

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

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Reduced Lamin A/C does Not Facilitate Cancer Cell Transendothelial Migration but Compromises Lung Metastasis

Cancers ◽

10.3390/cancers13102383 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2383

Author(s):

Francesco Roncato ◽

Ofer Regev ◽

Sara W. Feigelson ◽

Sandeep Kumar Yadav ◽

Lukasz Kaczmarczyk ◽

...

Keyword(s):

Cancer Cells ◽

Lung Metastasis ◽

Metastatic Cancer ◽

Nuclear Lamina ◽

Transendothelial Migration ◽

Soft Agar ◽

Lamin A ◽

And Migration

The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.

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Production and Function of the Cytoplasmic Deproteinized Relaxed Circular DNA of Hepadnaviruses

Journal of Virology ◽

10.1128/jvi.01921-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 387-396 ◽

Cited By ~ 88

Author(s):

Haitao Guo ◽

Richeng Mao ◽

Timothy M. Block ◽

Ju-Tao Guo

Keyword(s):

Dna Polymerase ◽

Structural Changes ◽

Core Protein ◽

Dnase I ◽

Dominant Negative ◽

Stable Cell Line ◽

Circular Dna ◽

Nuclear Localization Signals ◽

Infected Cells

ABSTRACT Removal of genome-bound viral DNA polymerase ought to be an essential step in the formation of hepadnavirus covalently closed circular DNA (cccDNA). We previously demonstrated that deproteinized (DP) relaxed circular DNA (rcDNA) of hepatitis B virus (HBV) existed in both the cytoplasm and nuclei of infected cells and the vast majority of cytoplasmic DP rcDNA was associated with DNase I-permeable nucleocapsids. In our efforts to investigate the role of the cytoplasmic DP rcDNA in cccDNA formation, we demonstrated that rcDNA deproteinization could occur in an endogenous DNA polymerase reaction with either virion-derived or intracellular nucleocapsids. As observed in the cytoplasm of virally infected cells, in vitro deproteinization requires the maturation of plus-strand DNA and results in changes in nucleocapsid structure that render the DP rcDNA susceptible to DNase I digestion. Remarkably, we found that the cytoplasmic DP rcDNA-containing nucleocapsids could be selectively immunoprecipitated with an antibody against the carboxyl-terminal peptide of HBV core protein and are associated with cellular nuclear transport receptors karyopherin-α and -β. Moreover, transfection of small interfering RNA targeting karyopherin-β1 mRNA or expression of a dominant-negative karyopherin-β1 in a stable cell line supporting HBV replication resulted in the accumulation of DP rcDNA in cytoplasm and reduction of nuclear DP rcDNA and cccDNA. Our results thus favor a hypothesis that completion of plus-strand DNA synthesis triggers the genomic DNA deproteinization and structural changes of nucleocapsids, which leads to the exposure of nuclear localization signals in the C terminus of core protein and mediates the nuclear transportation of DP rcDNA via interaction with karyopherin-α and -β.

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Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

Journal of General Virology ◽

10.1099/vir.0.83633-0 ◽

2008 ◽

Vol 89 (5) ◽

pp. 1212-1219 ◽

Cited By ~ 30

Author(s):

Hai-Jun Xu ◽

Zhang-Nv Yang ◽

Jin-Fang Zhao ◽

Cai-Hong Tian ◽

Jun-Qing Ge ◽

...

Keyword(s):

Bombyx Mori ◽

Cultured Cells ◽

Virus Production ◽

Core Gene ◽

Virus Protein ◽

Infected Cells ◽

Budded Virus ◽

Larval Bioassay

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

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Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00029-13 ◽

2013 ◽

Vol 57 (6) ◽

pp. 2761-2767 ◽

Cited By ~ 29

Author(s):

Arun Kapoor ◽

Ran He ◽

Rajkumar Venkatadri ◽

Michael Forman ◽

Ravit Arav-Boger

Keyword(s):

Stem Cell ◽

Cancer Stem Cell ◽

Human Cytomegalovirus ◽

Wnt Pathway ◽

Critical Role ◽

Wnt Signaling Pathway ◽

Negative Regulator ◽

Infected Cells ◽

Hcmv Replication

ABSTRACTInfection with human cytomegalovirus (HCMV) continues to be a threat for pregnant women and immunocompromised hosts. Although limited anti-HCMV therapies are available, development of new agents is desired. The Wnt signaling pathway plays a critical role in embryonic and cancer stem cell development and is targeted by gammaherpesviruses, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV). HCMV infects stem cells, including neural progenitor cells, during embryogenesis. To investigate the role of Wnt in HCMV replicationin vitro, we tested monensin, nigericin, and salinomycin, compounds that inhibit cancer stem cell growth by modulating the Wnt pathway. These compounds inhibited the replication of HCMV Towne and a clinical isolate. Inhibition occurred prior to DNA replication but persisted throughout the full replication cycle. There was a significant decrease in expression of IE2, UL44, and pp65 proteins. HCMV infection resulted in a significant and sustained decrease in expression of phosphorylated and total lipoprotein receptor-related protein 6 (pLRP6 and LRP6, respectively), Wnt 5a/b, and β-catenin and a modest decrease in Dvl2/3, while levels of the negative regulator axin 1 were increased. Nigericin decreased the expression of pLRP6, LRP6, axin 1, and Wnt 5a/b in noninfected and HCMV-infected cells. For all three compounds, a correlation was found between expression levels of Wnt 5a/b and axin 1 and HCMV inhibition. The decrease in Wnt 5a/b and axin 1 expression was more significant in HCMV-infected cells than noninfected cells. These data illustrate the complex effects of HCMV on the Wnt pathway and the fine balance between Wnt and HCMV, resulting in abrogation of HCMV replication. Additional studies are required to elucidate how HCMV targets Wnt for its benefit.

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The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

Journal of Virology ◽

10.1128/jvi.01967-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2065-2078 ◽

Cited By ~ 38

Author(s):

Eva Maria Borst ◽

Karen Wagner ◽

Anne Binz ◽

Beate Sodeik ◽

Martin Messerle

Keyword(s):

Human Cytomegalovirus ◽

Viral Genome ◽

Essential Role ◽

Unit Length ◽

Viral Dna ◽

Subnuclear Localization ◽

Viral Genomes ◽

Nuclear Egress ◽

C Terminus ◽

Infected Cells

ABSTRACT Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

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Activation of the NF-κB Pathway in Human Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major Immediate-Early Promoter

Journal of Virology ◽

10.1128/jvi.78.9.4498-4507.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4498-4507 ◽

Cited By ~ 98

Author(s):

Ian B. DeMeritt ◽

Liesl E. Milford ◽

Andrew D. Yurochko

Keyword(s):

Human Cytomegalovirus ◽

Time Course ◽

Hcmv Infection ◽

Dominant Negative ◽

Immediate Early ◽

Primary Role ◽

Early Promoter ◽

Ikk Complex ◽

Infected Cells ◽

Major Immediate Early Promoter

ABSTRACT We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-κB. Here, we investigate the mechanism for the HCMV-induced NF-κB activation and the role that the induced NF-κB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-κB, the IκB-superrepressor, we demonstrated that active NF-κB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-κB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-κB, IκBα and IκBβ. Because the IκB kinases (IKKs) regulate the degradation of the IκBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-κB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-κB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.

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Brd4 Regulation of Papillomavirus Protein E2 Stability

Journal of Virology ◽

10.1128/jvi.00674-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8683-8692 ◽

Cited By ~ 24

Author(s):

Gang Zheng ◽

Michal-Ruth Schweiger ◽

Gustavo Martinez-Noel ◽

Leon Zheng ◽

Jennifer A. Smith ◽

...

Keyword(s):

Transcriptional Activation ◽

Activation Function ◽

Dominant Negative ◽

Genome Maintenance ◽

E2 Protein ◽

E3 Ligases ◽

Protein Levels ◽

Dividing Cells ◽

Cullin 3 ◽

Infected Cells

ABSTRACT The papillomavirus (PV) E2 protein is an important regulator of the viral life cycle. It has diverse roles in viral transcription, DNA replication, and genome maintenance. Our laboratory has previously identified the cellular bromodomain protein Brd4 as a key interacting partner of E2. Brd4 mediates the transcriptional activation function of E2 and plays an important role in viral genome maintenance in dividing cells. E2 interacts with the C-terminal domain (CTD) of Brd4, and the CTD functions in a dominant-negative manner through binding E2 and interfering with E2's interaction with the full-length Brd4 protein. Previous studies have shown that PV E2 proteins are short lived; however, the mechanisms regulating their stability and degradation have not yet been well established. In this study, we explored the role of Brd4 in the regulation of bovine PV 1 (BPV1) and human PV 16 (HPV16) E2 stability. Expression of the Brd4 CTD dramatically increases E2 levels. Both BPV1 E2 and HPV16 E2 are regulated by ubiquitylation, and Brd4 CTD expression blocks this ubiquitylation, thus stabilizing the E2 protein. Furthermore, we have identified the cullin-based E3 ligases and specifically cullin-3 as potential components of the ubiquitylation machinery that targets both BPV1 and HPV16 E2 for ubiquitylation. Expression of the Brd4 CTD blocks the interaction between E2 and the cullin-3 complex. In addition to Brd4's role in mediating E2 transcription and genome tethering activities, these data suggest a potential role for Brd4 in regulating E2 stability and protein levels within PV-infected cells.

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