Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence and Western blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.
The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.
23The cellular protein SPOC1 (survival time-associated PHD finger protein in ovarian cancer 24 1) acts as a regulator of chromatin structure and DNA damage response. It binds 25H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting 26 corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified 27 SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is 28 antagonized by E1B-55K/E4orf6-dependent proteasomal degradation. Here, we 29 demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated 30 during the early phase of HCMV replication. We show that expression of the immediate-31 early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we 32 discovered that during later stages of infection SPOC1 is downregulated in a GSK-3β-33 dependent manner. We provide evidence that SPOC1 overexpression severely were known to mediate intrinsic immunity against HCMV. In this study, we identify the 52 chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that 53 pre-existing high SPOC1 protein levels mediate a silencing of HCMV gene expression via 54 a specific association with an important viral cis-regulatory element, the major immediate-55 early promoter. Since SPOC1 expression varies between cell-types, this factor may play 56 an important role in the tissue-specific defense against HCMV. 57
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