Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes

LaFemina, Robert L.; Pizzorno, Marie C.; Mosca, Joseph D.; Hayward, Gary S.

doi:10.1016/0042-6822(89)90201-8

Cited by 96 publications

(118 citation statements)

References 43 publications

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“…First, IE1 CORE is described as an elongated molecule consisting of a single contiguous domain with conserved residues distributed evenly over the entire surface (30). Second, IE1 CORE is hypersensitive to mutational interventions (36)(37)(38). Third, further attempts to narrow down the binding region by mutations and deletions failed.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies identified a central large core region of IE1 as being sufficient for interaction with PML; however, further attempts to narrow down the binding region by using mutations and deletions failed (29,30,(34)(35)(36)(37)(38). Indeed, the crystal structure of the rhesus cytomegalovirus IE1 CORE , which was recently solved by our group, provided strong evidence that IE1 CORE consists of a single contiguous domain which is very sensitive to deletion mutagenesis (30).…”

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confidence: 99%

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The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

Schilling

Scherer

Reuter

et al. 2017

J Virol

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PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML; however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays, demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner, since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As 2 O 3 -mediated hyperSUMOylation of K160, thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil-mediated PML dimerization, we propose that IE1 affects PML autoSUMOylation either by directly abrogating PML E3 ligase function or by preventing access to SUMO sites. Thus, our data suggest a novel mechanism for how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation. IMPORTANCEThe human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by subnuclear structures termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism for how IE1 antagonizes PML NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility of developing small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.KEYWORDS PML nuclear bodies, human cytomegalovirus, immediate early 1, intrinsic immunity, nuclear domain 10, sumoylation P romyelocytic leukemia protein (PML) is known to act as organizer of subnuclear, matrix-associated structures which can be detected as spherical dots of approximately 0.1 to 1.0 m (1). These structures, termed PML nuclear bodies (NBs) or nuclear domain 10 (ND10), are multiprotein complexes consisting of more than 160 proteins.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

Schilling

Scherer

Reuter

et al. 2017

J Virol

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“…The establishment of the A1b and A45 cell lines has been described previously (25,26). In transfected cells expressing IE-pp86 alone, the viral gene was inserted in the pMam-neo plasmid under the control of the dexamethasone-inducible Moloney murine tumor virus-long terminal repeat promoter.…”

Section: Cell Linesmentioning

confidence: 99%

“…ϩ mRNAs from peripheral blood monocytes were purified using Dynabeads oligo(dT) 25 (Dynal, Great Neck, NY). cDNAs were prepared by random hexamer priming of 100 ng of poly(A) ϩ mRNA.…”

Section: Poly(a)mentioning

confidence: 99%

Modulation of HLA-G Antigens Expression by Human Cytomegalovirus: Specific Induction in Activated Macrophages Harboring Human Cytomegalovirus Infection

Onno

Pangault

Friec

et al. 2000

The Journal of Immunology

154

115

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After infection, human CMV (HCMV) establishes a latent and persistent infection in immature myeloid progenitors and peripheral blood monocytes. Completion of the HCMV life cycle is possible upon maturation of monocytes to tissue macrophages and under permissive circumstances, e.g., immunosuppression. We investigated the hypothesis that HLA-G molecules could be induced during HCMV reactivation in activated macrophages to favor virus dissemination. In this study, we provide evidence that HLA-G Ags are produced during viral reactivation in macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. While HLA-G surface expression is up-regulated, classical MHC-I molecules are partially down-regulated by HCMV. In vivo, bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also express HLA-G molecules. The direct correlation between HLA-G Ag induction and HCMV infection was confirmed in U-373 MG astrocytoma cells. Soluble HLA-G expression is stimulated upon HCMV infection, and this modulation depends on the cooperative action of the two immediate-early-1 pp72 and immediate-early-2 pp86 products. Because HLA-G transcription is active in macrophages and U-373 MG astrocytoma cells, it is likely that the modulation of HLA-G protein expression during HCMV replication occurs at a post-transcriptional level. Our data suggest that induction of HLA-G molecules could be an additional mechanism that helps HCMV to subvert host defenses.

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“…The protein accumulates primarily in the nucleus of cells and associates with metaphase chromosomes (LaFemina et al, 1989). Although IE-1 transcript levels peak between 5 and 8 h post-infection, protein products continue to accumulate throughout infection (Stenberg et al, 1989).…”

mentioning

confidence: 99%

Monoclonal Antibody E-13 (M-810) to Human Cytomegalovirus Recognizes an Epitope Encoded by Exon 2 of the Major Immediate Early Gene

Mazeron

Jahn

Plachter

1992

Journal of General Virology

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Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes

Cited by 96 publications

References 43 publications

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

Modulation of HLA-G Antigens Expression by Human Cytomegalovirus: Specific Induction in Activated Macrophages Harboring Human Cytomegalovirus Infection

Monoclonal Antibody E-13 (M-810) to Human Cytomegalovirus Recognizes an Epitope Encoded by Exon 2 of the Major Immediate Early Gene

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