Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

Wagenknecht, Nadine; Reuter, Nina; Scherer, Myriam; Reichel, Anna; Müller, Regina; Stamminger, Thomas

doi:10.3390/v7062751

Cited by 66 publications

(78 citation statements)

References 61 publications

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“…On the other hand, the selective antagonization of individual PML-NB components by gammaherpesviruses may reflect that these viruses utilize specific functions of the cellular PML-NB repression machinery to promote latency as the default outcome of infection. So far, this appears to be unique to the gammaherpesviruses, since our recent studies on the role of PML-NBs during human cytomegalovirus latency do not reveal a major contribution of PML, Sp100, or hDaxx to the establishment of latent infections (21).…”

Section: Viral Pml-nb Antagonistsmentioning

confidence: 95%

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

Scherer

Stamminger

2016

J Virol

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153

168

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Research in the last 2 decades has demonstrated that a specific organelle of the cell nucleus, termed PML nuclear body (PML-NB) or nuclear domain 10 (ND10), is frequently modified during viral infection. This correlates with antagonization of a direct repressive function of individual PML-NB components, such as the PML, hDaxx, Sp100, or ATRX protein, that are able to act as cellular restriction factors. Recent studies now reveal an emerging role of PML-NBs as coregulatory structures of both type I and type II interferon responses. This emphasizes that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to compromise intrinsic antiviral defense and innate immune responses. P romyelocytic leukemia (PML) protein, a member of the tripartite motif (TRIM) protein family, is the key component of subnuclear structures known as PML nuclear bodies (PML-NBs) or nuclear domains 10 (ND10). PML-NBs are dynamic foci that consist of numerous permanently or transiently associated proteins and, consequently, have been implicated in the regulation of diverse cellular functions, including the cell cycle, apoptosis, senescence, stress, and DNA damage responses (reviewed in reference 1). Although the underlying biochemical function of these subnuclear structures is still unclear, three models can be found in the literature, proposing PML-NBs to be nuclear protein depots, sites of nuclear activities (e.g., transcription), or hotspots for posttranslational modifications. In particular, since nearly all PMLassociated proteins are modified by SUMO and SUMOylation of PML is essential for the integrity of PML-NBs, these structures may form "catalytic surfaces" for SUMOylation (1). This is of importance since recent studies suggest that the SUMO pathway is required for the regulation of innate immune signaling and intrinsic immunity during viral infection (reviewed in reference 2). The observation that PML-NBs are targeted and modified by many viruses during infection set off a longstanding debate as to whether PML-NBs exert a pro-or antiviral function and led to a fruitful area of virology research over the past 20 years. Interestingly, these studies revealed that viruses, even representatives of the same virus family, trigger diverse modifications of PML-NBs during infection, ranging from proteasomal degradation of NB components by herpes simplex virus type 1 (HSV-1), to dispersal of PML-NBs by human cytomegalovirus (HCMV), to a rearrangement of PML-NB foci into nuclear track-like structures by adenoviruses or a relocalization of PML into cytoplasmic bodies by HIV-1 (3-5). While there are a few examples of viral factors that undergo interactions with PML-NBs in order to exploit these structures for the benefit of the virus, the main body of evidence supports a role of PML-NBs as components of the antiviral defense against a variety of DNA and RNA viruses (reviewed in reference 3). PML-NBs AND INTRINSIC IMMUNITYThe role of PML-NBs in intrinsic immunity, which represents the first line of intracellular defense agains...

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Section: Viral Pml-nb Antagonistsmentioning

confidence: 95%

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

Scherer

Stamminger

2016

J Virol

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153

168

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“…The prokaryotic expression plasmid encoding GST-tagged IE1-L174P was constructed by PCR using pHM3260 as the template and primers 5=IE1-GEX-Bam and 3=IE1_Xho1, followed by ligation into pGEX-6P-1 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) via BamHI and XhoI. The lentiviral pLVX-shRNA1-based vectors encoding control short hairpin RNA (shRNA) and PML shRNA were described previously (48). Lentiviral vectors that are based on pLVX-Tight-Puro and encode IE1 or IE1 1-382, both fused to a FLAG-tag, were generated by PCR amplification with primer pairs 5=Flag_IE1_BamHI and 3=IE1_EcoRI_stop or 5=Flag_IE1_BamHI and 3=IE1aa382_EcoRI along with pHM494 as the template, followed by insertion into pLVX-Tight-Puro via BamHI and EcoRI.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses

Scherer

Otto

Stump

et al. 2016

J Virol

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PML is the organizer of cellular structures termed nuclear domain 10 (ND10) or PML-nuclear bodies (PML-NBs) that act as key mediators of intrinsic immunity against human cytomegalovirus (HCMV) and other viruses. The antiviral function of ND10 is antagonized by viral regulatory proteins such as the immediate early protein IE1 of HCMV. IE1 interacts with PML through its globular core domain (IE1 CORE ) and induces ND10 disruption in order to initiate lytic HCMV infection. Here, we investigate the consequences of a point mutation (L174P) in IE1 CORE , which was shown to abrogate the interaction with PML, for lytic HCMV infection. We found that a recombinant HCMV encoding IE1-L174P displays a severe growth defect similar to that of an IE1 deletion virus. Bioinformatic modeling based on the crystal structure of IE1 CORE suggested that insertion of proline into the highly alpha-helical domain severely affects its structural integrity. Consistently, L174P mutation abrogates the functionality of IE1 CORE and results in degradation of the IE1 protein during infection. In addition, our data provide evidence that IE1 CORE as expressed by a recombinant HCMV encoding IE1 1-382 not only is required to antagonize PML-mediated intrinsic immunity but also affects a recently described function of PML in innate immune signaling. We demonstrate a coregulatory role of PML in type I and type II interferon-induced gene expression and provide evidence that upregulation of interferon-induced genes is inhibited by IE1 CORE . In conclusion, our data suggest that targeting PML by viral regulatory proteins represents a strategy to antagonize both intrinsic and innate immune mechanisms. IMPORTANCEPML nuclear bodies (PML-NBs), which represent nuclear multiprotein complexes consisting of PML and additional proteins, represent important cellular structures that mediate intrinsic resistance against many viruses, including human cytomegalovirus (HCMV). During HCMV infection, the major immediate early protein IE1 binds to PML via a central globular domain (IE1 CORE ), and we have shown previously that this is sufficient to antagonize intrinsic immunity. Here, we demonstrate that modification of PML by IE1 CORE not only abrogates intrinsic defense mechanisms but also attenuates the interferon response during infection. Our data show that PML plays a novel coregulatory role in type I as well as type II interferon-induced gene expression, which is antagonized by IE1 CORE . Importantly, our finding supports the view that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to inhibit both intrinsic and innate immune defense mechanisms. H uman cytomegalovirus (HCMV), a member of the ␤-subgroup of herpesviruses, is a widespread human pathogen of high clinical relevance that can cause life-threatening diseases in newborns and people with compromised immune system such as AIDS, transplantation, or malignancy patients. The lytic replication cycle of HCMV is characterized by three sequential phases of viral gene expression, te...

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“…Therefore, we constructed a recombinant virus TB40E/IE2eYFP-delUS28 which lacks the US28 gene and possesses an eYFP-tagged IE2 enabling detection of lytically-infected cells. The TB40E/IE2eYFP-delUS28 and the previously described TB40E/IE2eYFP [50] viruses allowed us to monitor CXCR4 surface expression in lytically-infected cells via Fluorescence-activated cell sorting (FACS) analysis. We infected primary human umbilical vein endothelial cells (HUVEC) at a multiplicity of infection (MOI) of 2 which yielded between 10 and 30% lytically-infected cells.…”

Section: Resultsmentioning

confidence: 99%

“…Infection experiments were performed with the recombinant viruses TB40E/IE2-eYFP [50] and TB40E/IE2eYFP-delUS28. Titration of the viral stocks was performed by IE1p72 fluorescence [67].…”

Section: Methodsmentioning

confidence: 99%

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28

et al. 2016

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BackgroundSome herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency.ResultsWe investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection.ConclusionsWe show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0154-x) contains supplementary material, which is available to authorized users.

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Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

Cited by 66 publications

References 61 publications

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28

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