SP100B is a repressor of gene expression

185

293

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.Herpes simplex virus type 1 (HSV-1) is a common human pathogen that causes recurrent infections through its ability to establish a latent state in sensory ganglia after primary epithelial infections (for a general review, see reference 58). Lytic HSV-1 infection is characterized by abundant transcription from the entire viral genome in a temporal cascade of immediate-early (IE), early, and late gene products. The IE gene products regulate the expression of later classes of viral genes. In contrast, lytic cycle genes are repressed during latency, and only the latency-associated transcripts (LATs; derived from a single locus that lies countersense to the IE gene encoding ICP0) are expressed in readily detectable amounts (8,55,69). The IE protein ICP0 is a RING finger E3 ubiquitin ligase (4) that is required for efficient entry into the lytic cycle and which can induce reactivation of latent or quiescent genomes (reviewed in references 12, 14, 15, 29-31, and 55). ICP0 influences many cellular pathways, and one of its most prominent activities is its ability to localize to and disrupt nuclear substructures known as PML nuclear bodies (also known as ND10) (reviewed in references 10, 14, 16, and 43). This disruption occurs through ICP0-induced degradation of PML (17), the key component of ND10 which is required for assembly of these structures (34, 76). HSV-1 mutants that fail to express ICP0 or that express mutant ICP0 proteins that lack RING finger activity are unable to disrupt ND10 or to degrade PML (4,11,17,44,45). Such mutants have a profound defect in HSV-1 gene expression after infection of limited-passage human fibroblasts (9,21,62,63).The strong correlation between the effects of ICP0 on ND10and its requirement for lytic virus infection prompted the hypothesis that ND10 might ...

Section: Icp0mentioning

confidence: 99%

Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1 Is Restricted by both PML and Sp100

Everett¹,

Parada²,

Gripon³

et al. 2008

185

293

“…The direct roles of Sp100 proteins in HCMV growth have yet to be addressed, but the Sp100 proteins have been suggested to function as transcription regulators for herpesviral genes. In herpes simplex virus type 1 (HSV-1) infection, the expression of IE genes is suppressed by Sp100B, Sp100-HMG, and Sp100C but not by Sp100A (21,34,35,52). Additionally, it is notable that Sp100B, Sp100-HMG, and Sp100C harbor the SAND domain (a DNA binding domain [6]) within their C-terminal regions.…”

mentioning

confidence: 99%

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

Kim

Lee

Kim

et al. 2011

The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.During the early stages of human cytomegalovirus (HCMV) infection, the 72-kDa immediate-early 1 (IE1 or IE72) protein targets the subnuclear structures referred to as PML nuclear bodies (NBs) (also known as nuclear domain 10 [ND10] or PML oncogenic domains [PODs]), in which input viral genomes are deposited and IE transcription occurs (22). However, the targeting of IE1 to PML NBs is transient, and subsequently, PML NBs are disrupted in an IE1-dependent manner and both IE1 and the components of PML NBs, including PML and Sp100 proteins, are relocalized into the nucleoplasm (4, 25, 27, 53). Several lines of evidence suggest that this early event promotes viral replication. The overexpression of PML conferred resistance to HCMV infection (3), and the analysis of IE1 mutants demonstrated that the ability of IE1 to disrupt PML NBs was correlated with its transactivation activity and efficient viral growth in cells transfected with HCMVbacterial artificial chromosome (BAC) DNA (30). In addition, the depletion of PML by RNA interference has been reported to promote viral replication efficiency (47). These results support the notion that PML NBs are intrinsic defense sites at which the epigenetic silencing of input viral DNA genome may take place and that the components of PML NBs perform antiviral roles against a variety of DNA and RNA vir...

“…Similarly, Sp100 also behaves as a transcriptional repressor via interaction with the heterochromatin protein HP1 (41,60,63). The fact that several ND10 components associate with potent repressors of gene expression gave rise to the idea of ND10 acting as sites of transcriptional repression.…”

mentioning

confidence: 99%

Evidence for a Role of the Cellular ND10 Protein PML in Mediating Intrinsic Immunity against Human Cytomegalovirus Infections

Tavalai

Papior

Rechter

et al. 2006

197

257

Several viruses, including human cytomegalovirus (HCMV), encode proteins that colocalize with a cellular subnuclear structure known as ND10. Since only viral DNA deposited at ND10 initiates transcription, ND10 structures were hypothesized to be essential for viral replication. On the other hand, interferon treatment induces an up-regulation of ND10 structures and viruses have evolved polypeptides that disperse the dot-like accumulation of ND10 proteins, suggesting that ND10 could also be part of an intrinsic defense mechanism. In order to obtain evidence for either a proviral or an antiviral function of ND10, we generated primary human fibroblasts with a stable, short interfering RNA-mediated knockdown (kd) of PML. In these cells, other ND10-associated proteins like hDaxx showed a diffuse nuclear distribution. Interestingly, we observed that HCMV infection induced the de novo formation of ND10-like hDaxx and Sp100 accumulations that colocalized with IE2 and were disrupted, in the apparent absence of PML, in an IE1-dependent manner during the first hours after infection. Furthermore, infection of PML-kd cells with wild-type HCMV at a low multiplicity of infection resulted in enhanced replication. In particular, a significantly increased plaque formation was detected, suggesting that more cells are able to support initiation of replication in the absence of PML. While there was no difference in viral DNA uptake between PML-kd and control cells, we observed a considerable increase in the number of immediate-early (IE) protein-positive cells, indicating that the depletion of PML augments the initiation of viral IE gene expression. These results strongly suggest that PML functions as part of an intrinsic immune mechanism against cytomegalovirus infections.In addition to the conventional innate and adaptive immune responses, it was recently recognized that complex organisms have evolved a set of constitutively expressed genes that are able to repress viral infections. These so-called intrinsic immune mechanisms involve the APOBEC3 class of cytidine deaminases as well as a large family of proteins termed the TRIM family (7, 52). We were interested in determining the role of the interferon-inducible TRIM19, also known as promyelocytic leukemia protein (PML), for human cytomegalovirus (HCMV) replication. PML is essential for the integrity of a cellular subnuclear structure, termed ND10, which has been shown to colocalize with herpesvirus DNA during infection (47).ND10 domains, also known as nuclear dots, PML nuclear bodies, or promyelocytic oncogenic domains, are spherical nuclear substructures which represent accumulations of multiple cellular proteins like Sp100, hDaxx, BLM, or SUMO-1 that require the PML protein for their formation (51). Since PML constitutes the defining component of ND10, loss of PML consequently leads to a dispersal of other ND10-associated proteins as observed in mouse PML-null fibroblasts (30,66,67). The PML protein was originally discovered in patients suffering from acute promyelocytic leukemia...