Evidence for a Dual Antiviral Role of the Major Nuclear Domain 10 Component Sp100 during the Immediate-Early and Late Phases of the Human Cytomegalovirus Replication Cycle

Abstract

In recent studies, the nuclear domain 10 (ND10) components PML and hDaxx were identified as cellular restriction factors that inhibit the initiation of human cytomegalovirus (HCMV) replication. The antiviral function of ND10, however, is antagonized by the IE1 protein, which induces ND10 disruption. Here we show that IE1 not only de-SUMOylates PML immediately upon infection but also directly targets Sp100. IE1 expression alone was sufficient to downregulate endogenous Sp100 independently of the presence of PML. Moreover, cotransfection experiments revealed that IE1 negatively interferes with the SUMOylation of all Sp100 isoforms. The modulation of Sp100 at immediate-early (IE) times of infection, indeed, seemed to have an in vivo relevance for HCMV replication, since knockdown of Sp100 resulted in more cells initiating the viral gene expression program. In addition, we observed that Sp100 was degraded in a proteasome-dependent manner at late times postinfection, suggesting that Sp100 may play an additional antiviral role during the late phase. Infection experiments conducted with Sp100 knockdown human foreskin fibroblasts (HFFs) confirmed this hypothesis: depletion of Sp100 resulted in augmented release of progeny virus particles compared to that from control cells. Consistent with this observation, we noted increased amounts of viral late gene products in the absence of Sp100. Importantly, this elevated late gene expression was not dependent on enhanced viral IE gene expression. Taken together, our data provide evidence that Sp100 is the first ND10-related factor identified that not only possesses the potential to restrict the initial stage of infection but also inhibits HCMV replication during the late phase.

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

“…In accordance with previous studies, PML was found to be partially deSUMOylated upon infection ( Fig. 1 A) (29,40). While most high-molecular-weight bands of PML disappeared, one presumably modified species of PML seemed to be resistant against IE1 action (Fig.…”

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

“…Furthermore, this complete loss of PML as a consequence of MHV-68 infection presumably also accounted for the simultaneously observed disappearance of the low-mobility isoforms of Sp100 (Fig. 1B, lane 2), comparable to the situation in herpes simplex virus type 1-infected cells (7) or after depletion of the defining ND10 factor in HFFs by short hairpin RNA-mediated knockdown of PML (12,36). Intriguingly, PML protein levels remained completely unaltered following infection with HVS (Fig.…”

“…Since sumoylation of PML is essential for the integrity of ND10, this leads to a dispersal of the subnuclear structure. Interestingly, we and others could recently show that IE1 can also directly affect Sp100 (20,36). In particular, at late times of the human CMV (HCMV) replicative cycle, a proteasomal degradation of Sp100 was observed; however, degradation was dependent on the additional presence of viral true late gene products (20,36).…”

“…To address this issue, primary HFFs were infected in parallel with recombinant GFP-expressing HVS and MHV-68 using high-multiplicity-of-infection (high-MOI) conditions, as verified by monitoring of GFP-positive cells (data not shown). Importantly, HFF cells were chosen for this purpose since ND10 bodies have already been extensively characterized in these cells in prior studies of our group (36,37,38). Then, 24 h after virus inoculation, cell extracts were harvested and analyzed for PML, Sp100, and hDaxx protein levels ( Fig.…”

“…HFFs with a small interfering RNA (siRNA)-mediated knockdown of PML (PML-kd; siPML2 cells), hDaxx (siDaxx1 cells), and Sp100 (siSp100 cells) and the respective control cells (designated vector or siC) were cultured in Dulbecco's minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and 5 g/ml puromycin (2,36,37). Primary rhesus fibroblasts (kindly provided by A. Kaur, Harvard Medical School, Southborough, MA), OMK cells, and Vero cells were cultivated in Dulbecco's minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and gentamicin.…”