Sabine Rechter

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0.

ABSTRACT Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.

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

ABSTRACT 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.

Nuclear Domain 10 Components Promyelocytic Leukemia Protein and hDaxx Independently Contribute to an Intrinsic Antiviral Defense against Human Cytomegalovirus Infection

ABSTRACT Infection with DNA viruses commonly results in the association of viral genomes with a cellular subnuclear structure known as nuclear domain 10 (ND10). Recent studies demonstrated that individual ND10 components, like hDaxx or promyelocytic leukemia protein (PML), mediate an intrinsic immune response against human cytomegalovirus (HCMV) infection, strengthening the assumption that ND10 components are part of a cellular antiviral defense mechanism. In order to further define the role of hDaxx and PML for HCMV replication, we generated either primary human fibroblasts with a stable, individual knockdown of PML or hDaxx (PML-kd and hDaxx-kd, respectively) or cells exhibiting a double knockdown. Comparative analysis of HCMV replication in PML-kd or hDaxx-kd cells revealed that immediate-early (IE) gene expression increased to a similar extent, regardless of which ND10 constituent was depleted. Since a loss of PML, the defining component of ND10, results in a dispersal of the entire nuclear substructure, the increased replication efficacy of HCMV in PML-kd cells could be a consequence of the dissociation of the repressor protein hDaxx from its optimal subnuclear localization. However, experiments using three different recombinant HCMVs revealed a differential growth complementation in PML-kd versus hDaxx-kd cells, strongly arguing for an independent involvement in suppressing HCMV replication. Furthermore, infection experiments using double-knockdown cells devoid of both PML and hDaxx illustrated an additional enhancement in the replication efficacy of HCMV compared to the single-knockdown cells. Taken together, our data indicate that both proteins, PML and hDaxx, mediate an intrinsic immune response against HCMV infection by contributing independently to the silencing of HCMV IE gene expression.

Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo

Cytomegalovirus infection is associated with severe disease in immunocompromised individuals. Current antiviral therapy faces several limitations. In a search of novel drug candidates, we describe here the anti-cytomegaloviral properties of two compounds of the chemical class of quinazolines, gefitinib (Iressa) and Ax7396 (RGB-315389). Both compounds showed strong inhibitory effects in vitro against human and animal cytomegaloviruses with IC(50)s in a low micromolar range. Cytotoxicity did not occur at these effective concentrations. The antiviral mode of action was based on the inhibition of protein kinase activity, mainly directed to a viral target kinase (UL97/M97) in addition to cellular target candidates. This was demonstrated by a high sensitivity of the respective protein kinases in vitro and by infection experiments with viral mutants carrying genomic alterations in the ORF UL97/M97 modulating viral drug sensitivity. In a guinea pig model, gefitinib showed inhibition of cytomegaloviral loads in blood and lung tissue. Importantly, the rate of mortality of infected animals was reduced by gefitinib treatment. In contrast to the in vitro data, Ax7396 showed no significant antiviral activity in a mouse model. Further in vivo analyses have to assess the potential use of gefitinib in the treatment of cytomegalovirus disease.

Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69-pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1-140/478-532 in pUL69 and 231-336 in pUL97). Further analysis of the immunoprecipitates by in vitro kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69-pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity.

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69.