Silencing of Human Cytomegalovirus Gene Expression Mediated by Components of PML Nuclear Bodies

2016 ◽  
pp. 175-196 ◽  
Author(s):  
Myriam Scherer ◽  
Nadine Wagenknecht ◽  
Nina Reuter ◽  
Thomas Stamminger
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Nuclear Bodies ◽  
Pml Nuclear Bodies

scholarly journals Analysis of the Functional Interchange between the IE1 and pp71 Proteins of Human Cytomegalovirus and ICP0 of Herpes Simplex Virus 1

2014 ◽  
Vol 89 (6) ◽  
pp. 3062-3075 ◽  
Author(s):  
Yongxu Lu ◽  
Roger D. Everett
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Human Cytomegalovirus ◽  
Nuclear Bodies ◽  
Herpes Simplex Virus 1 ◽  
Pml Nuclear Bodies ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHuman cytomegalovirus (HCMV) immediate early protein IE1 and the tegument protein pp71 are required for efficient infection. These proteins have some functional similarities with herpes simplex virus 1 (HSV-1) immediate early protein ICP0, which stimulates lytic HSV-1 infection and derepresses quiescent HSV-1 genomes. All three proteins counteract antiviral restriction mediated by one or more components of promyelocytic leukemia (PML) nuclear bodies, and IE1 and pp71, acting together, almost completely complement ICP0 null mutant HSV-1. Here, we investigated whether ICP0 might substitute for IE1 or pp71 during HCMV infection. Using human fibroblasts that express ICP0, IE1, or pp71 in an inducible manner, we found that ICP0 stimulated replication of both wild-type (wt) and pp71 mutant HCMV while IE1 increased wt HCMV plaque formation and completely complemented the IE1 mutant. Although ICP0 stimulated IE2 expression from IE1 mutant HCMV and increased the number of IE2-positive cells, it could not compensate for IE1 in full lytic replication. These results are consistent with previous evidence that both IE1 and IE2 are required for efficient HCMV gene expression, but they also imply that IE2 functionality is influenced specifically by IE1, either directly or indirectly, and that IE1 may include sequences that have HCMV-specific functions. We discovered a mutant form of IE1 (YL2) that fails to stimulate HCMV infection while retaining 30 to 80% of the activity of the wt protein in complementing ICP0 null mutant HSV-1. It is intriguing that the YL2 mutation is situated in the region of IE1 that is shared with IE2 and which is highly conserved among primate cytomegaloviruses.IMPORTANCEHerpesvirus gene expression can be repressed by cellular restriction factors, one group of which is associated with structures known as ND10 or PML nuclear bodies (PML NBs). Regulatory proteins of several herpesviruses interfere with PML NB-mediated repression, and in some cases their activities are transferrable between different viruses. For example, the requirement for ICP0 during herpes simplex virus 1 (HSV-1) infection can be largely replaced by ICP0-related proteins expressed by other alphaherpesviruses and even by a combination of the unrelated IE1 and pp71 proteins of human cytomegalovirus (HCMV). Here, we report that ICP0 stimulates gene expression and replication of wt HCMV but cannot replace the need for IE1 during infection by IE1-defective HCMV mutants. Therefore, IE1 includes HCMV-specific functions that cannot be replaced by ICP0.

scholarly journals Epstein–Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments

2006 ◽  
Vol 87 (5) ◽  
pp. 1133-1137 ◽  
Author(s):  
Wolfgang Amon ◽  
Robert E. White ◽  
Paul J. Farrell
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Nuclear Bodies ◽  
Lytic Cycle ◽  
Promyelocytic Leukaemia ◽  
Late Gene ◽  
Barr Virus ◽  
Pml Nuclear Bodies ◽  
Epstein Barr

Epstein–Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain–enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

scholarly journals Human Cytomegalovirus Protein pp71 Induces Daxx SUMOylation

2009 ◽  
Vol 83 (13) ◽  
pp. 6591-6598 ◽  
Author(s):  
Jiwon Hwang ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Protein Function ◽  
Viral Infections ◽  
Biological Significance ◽  
Qualitative Change ◽  
Nuclear Bodies ◽  
Cellular Processes ◽  
Unknown Protein ◽  
Lysine Residues

ABSTRACT Proteins that participate in a diverse array of cellular processes can be modified covalently and reversibly on lysine residues by the small ubiquitin-like modifier proteins termed SUMOs. In some instances, such modification profoundly affects protein function, but the biological significance of many SUMOylation events remains unknown. Protein SUMOylation is modulated during many viral infections. Here we demonstrate that the human cytomegalovirus (HCMV) pp71 protein promotes the SUMOylation of its cellular substrate, Daxx. A component of promyelocytic leukemia nuclear bodies, Daxx is a transcriptional corepressor that silences the expression of viral immediate-early (IE) genes at the start of both lytic and quiescent HCMV infections. pp71 is a tegument component delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process. Here we report that pp71 also substantially increases the basal level of SUMOylated Daxx observed in cells. To date, consequences of Daxx SUMOylation have not been observed for cellular promoters, and we detected no qualitative change in viral IE gene expression in the absence of pp71-induced Daxx SUMOylation. Thus, while pp71 enhances the basal level of SUMOylated Daxx, the role that this modification plays in regulating Daxx activity in uninfected or HCMV-infected cells remains an enigma.

scholarly journals Binding STAT2 by the Acidic Domain of Human Cytomegalovirus IE1 Promotes Viral Growth and Is Negatively Regulated by SUMO

2008 ◽  
Vol 82 (21) ◽  
pp. 10444-10454 ◽  
Author(s):  
Yong Ho Huh ◽  
Young Eui Kim ◽  
Eui Tae Kim ◽  
Jung Jin Park ◽  
Moon Jung Song ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Mutant Virus ◽  
Nuclear Bodies ◽  
Hydrophobic Region ◽  
Viral Growth ◽  
Interferon Stimulated Genes ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Pml Nuclear Bodies ◽  
Fold Reduction

ABSTRACT The human cytomegalovirus (HCMV) 72-kDa immediate-early 1 (IE1) protein is thought to modulate cellular antiviral functions impacting on promyelocytic leukemia (PML) nuclear bodies and signal transducer and activator of transcription (STAT) signaling. IE1 consists of four distinct regions: an amino-terminal region required for nuclear localization, a large central hydrophobic region responsible for PML targeting and transactivation activity, an acidic domain, and a carboxyl-terminal chromatin tethering domain. We found that the acidic domain of IE1 is required for binding to STAT2. A mutant HCMV encoding IE1(Δ421-475) with the acidic domain deleted was generated. In mutant virus-infected cells, IE1(Δ421-475) failed to bind to STAT2. The growth of mutant virus was only slightly delayed at a high multiplicity of infection (MOI) but was severely impaired at a low MOI with low-level accumulation of viral proteins. When cells were pretreated with beta interferon, the mutant virus showed an additional 1,000-fold reduction in viral growth, even at a high MOI, compared to the wild type. The inhibition of STAT2 loading on the target promoter upon infection was markedly reduced with mutant virus. Furthermore, sumoylation of IE1 at this acidic domain was found to abolish the activity of IE1 to bind to STAT2 and repress the interferon-stimulated genes. Our results provide genetic evidence that IE1 binding to STAT2 requires the 55-amino-acid acidic domain and promotes viral growth by interfering with interferon signaling and demonstrate that this viral activity is negatively regulated by a cellular sumoylation pathway.

scholarly journals Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells

2007 ◽  
Vol 81 (18) ◽  
pp. 10123-10136 ◽  
Author(s):  
George Sourvinos ◽  
Nina Tavalai ◽  
Anja Berndt ◽  
Demetrios A. Spandidos ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Spatial Organization ◽  
Human Fibroblasts ◽  
Nuclear Bodies ◽  
Immediate Early ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Pml Nuclear Bodies ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise colocalizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.

scholarly journals Role for Centromeric Heterochromatin and PML Nuclear Bodies in the Cellular Response to Foreign DNA

2006 ◽  
Vol 26 (7) ◽  
pp. 2583-2594 ◽  
Author(s):  
Cleo L. Bishop ◽  
Michal Ramalho ◽  
Nachiket Nadkarni ◽  
Wing May Kong ◽  
Christopher F. Higgins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgene Expression ◽  
Nuclear Bodies ◽  
Luciferase Reporter ◽  
Centromeric Heterochromatin ◽  
Type I ◽  
Cell Nuclei ◽  
Pml Nuclear Bodies ◽  
Activation Of Transcription ◽  
Spatial Positioning

ABSTRACT Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-α). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment.

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

2016 ◽  
Vol 91 (4) ◽  
Author(s):  
Eva-Maria Schilling ◽  
Myriam Scherer ◽  
Nina Reuter ◽  
Johannes Schweininger ◽  
Yves A. Muller ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
De Novo ◽  
Tight Binding ◽  
Coiled Coil ◽  
Nuclear Bodies ◽  
Intrinsic Immunity ◽  
Pml Nuclear Bodies ◽  
Early Protein

ABSTRACT 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 As2O3-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. IMPORTANCE The 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.

scholarly journals Human Papillomavirus Infectious Entry and Trafficking Is a Rapid Process

2015 ◽  
Vol 89 (17) ◽  
pp. 8727-8732 ◽  
Author(s):  
Justyna Broniarczyk ◽  
Paola Massimi ◽  
Martina Bergant ◽  
Lawrence Banks
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Papillomavirus ◽  
Intracellular Transport ◽  
Nuclear Bodies ◽  
Viral Gene ◽  
Rapid Process ◽  
Pml Nuclear Bodies ◽  
Infectious Entry ◽  
In Cells

ABSTRACTPrevious studies have indicated that human papillomavirus (HPV) infectious entry is slow, requiring many hours after initial infection for the virus to gain entry into the nucleus. However, intracellular transport pathways typically are very rapid, and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem to be at odds with a normal viral infection. Using synchronized cell populations, we show that HPV trafficking can be a rapid process. In cells that are infected in the late S-early G2/M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8 h postinfection. Likewise, reporter DNA can be visualized within the nucleus in conjunction with PML nuclear bodies 1 h to 2 h postinfection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry.IMPORTANCEHPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study, we show that HPV trafficking in the cell actually is very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

scholarly journals Small ubiquitin-related modifier (SUMO) pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments

2013 ◽  
Vol 94 (6) ◽  
pp. 1373-1384 ◽  
Author(s):  
Myriam Scherer ◽  
Nina Reuter ◽  
Nadine Wagenknecht ◽  
Victoria Otto ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Regulatory Protein ◽  
Nuclear Bodies ◽  
Antiviral Resistance ◽  
Sumo Conjugation ◽  
Pml Nuclear Bodies ◽  
Hcmv Replication ◽  
Simplex Virus

Recent studies have suggested that the small ubiquitin-related modifier (SUMO) conjugation pathway may play an important role in intrinsic antiviral resistance and thus for repression of herpesviral infections. In particular, it was shown that the herpes simplex virus type-1 regulatory protein ICP0 acts as a SUMO-targeted ubiquitin ligase (STUbL), inducing the widespread degradation of SUMO-conjugated proteins during infection. As the IE1 protein of human cytomegalovirus (HCMV) is known to mediate a de-SUMOylation of PML, we investigated whether HCMV uses a similar mechanism to counteract intrinsic antiviral resistance. We generated primary human fibroblasts stably expressing FLAG-SUMO-1 or FLAG-SUMO-3 and analysed the SUMOylation pattern after HCMV infection or isolated IE1 expression. However, Western blot experiments did not reveal a global loss of SUMO conjugates, either in HCMV-infected or in IE1-expressing cells, arguing against a function of IE1 as an STUbL. Interestingly, we observed that FLAG-SUMO-1 and FLAG-SUMO-3, subsequent to IE1-mediated promyelocytic leukemia protein (PML) de-SUMOylation and the consequent disruption of PML nuclear bodies, were recruited into viral replication compartments. This raised the question of whether FLAG-SUMO-1/3 might promote HCMV replication. Intriguingly, overexpression of FLAG-SUMO-1/3 enhanced accumulation of viral DNA, which correlated with an increase in viral replication and in virus particle release. Together, these data indicate that HCMV, in contrast to other herpesviruses, has evolved subtle mechanisms enabling it to utilize the SUMO conjugation pathway for its own benefit, resulting in an overall positive effect of SUMO conjugation for HCMV replication.

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