Human Cytomegalovirus Infection Causes Degradation of Sp100 Proteins That Suppress Viral Gene Expression

In recent studies, the nuclear domain 10 (ND10) components PML, Sp100, human Daxx (hDaxx), and ATRX were identified to be cellular restriction factors that are able to inhibit the replication of several herpesviruses. The antiviral function of ND10, however, is antagonized by viral effector proteins by a variety of strategies, including degradation of PML or relocalization of ND10 proteins. In this study, we analyzed the interplay between infection with herpesvirus saimiri (HVS), the prototypic rhadinovirus, and cellular defense by ND10. In contrast to other herpesviruses, we found that HVS specifically degraded the cellular ND10 component Sp100, whereas other factors like PML or hDaxx remained intact. We could further identify the ORF3 tegument protein of HVS, which shares homology with the cellular formylglycinamide ribotide amidotransferase (FGARAT) enzyme, to be the viral factor that induces the proteasomal degradation of Sp100. Interestingly, recent studies showed that the ORF3-homologous proteins ORF75c of murine gammaherpesvirus 68 and BNRF-1 of Epstein-Barr virus modulate the ND10 proteins PML and ATRX, respectively, suggesting that the ND10 targets of viral FGARAT-homologous proteins diversified during evolution. Furthermore, a virus with the ORF3 deletion was efficiently complemented in Sp100-depleted cells, indicating that Sp100 is able to inhibit HVS in the absence of antagonistic mechanisms. In contrast, we observed that PML, which was neither degraded nor redistributed after HVS infection, strongly restricted both wild-type HVS and virus with the ORF3 deletion. Thus, HVS may lack a factor that efficiently counteracts the repressive function of PML, which may foster latency as the outcome of infection.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Herpesvirus Saimiri Antagonizes Nuclear Domain 10-Instituted Intrinsic Immunity via an ORF3-Mediated Selective Degradation of Cellular Protein Sp100

Full

Reuter

Zielke

et al. 2012

“…Covalent and noncovalent interactions of PML with the small ubiquitin-like modifier (SUMO) protein are essential for the assembly and maintenance of these protein complexes (17)(18)(19)(20). Besides PML, the ND10 proteins Sp100, hDaxx, and ATRX function as cellular restriction factors and act cooperatively in order to restrict HCMV infection (21)(22)(23)(24)(25)(26)(27)(28)). An interplay between intrinsic and innate immune mechanisms has been reported, since several cellular restriction factors, including PML and Sp100, are upregulated after interferon (IFN) treatment, which enhances their antiviral activity (29)(30)(31).…”

mentioning

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

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...

“…Most critical in this respect are the 72-kDa IE1 (synonym, IE72) and the 86-kDa IE2 (IE86) nuclear phosphoproteins, both originating from the abundantly transcribed "major IE" (MIE) gene loci UL122 and -123. IE1 derepresses early gene promoters by antagonizing PML-, Sp100-, Daxx/ATRX-, and HDAC3-mediated histone deacetylation (34,50,52,64). It is required, however, only at low multiplicities of infection (MOI), because at high MOI, the increased abundance of incoming viral tegument proteins compensates for the loss of IE1 (20).…”

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confidence: 99%

Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

Oduro

Uecker

Hagemeier

et al. 2012

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G 0 /G 1 phase of the cell division cycle. S/G 2 cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G 2 -specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G 1 was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.