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PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate–early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells., publishedVersion

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

Nuclear egress is a common herpesviral process regulating nucleocytoplasmic capsid release. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that regulates multicomponent assembly with NEC-associated proteins and capsids. Recently, NEC crystal structures were resolved for α-, β- and γ-herpesviruses, revealing profound structural conservation, which was not mirrored, however, by primary sequence and binding properties. The NEC binding principle is based on hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. So far, pUL50 has been considered as the major kinase-interacting determinant and massive phosphorylation of pUL50-pUL53 was assigned to NEC formation and functionality. Here, we addressed the question of phenotypical changes of ORF-UL50-mutated HCMVs. Surprisingly, our analyses did not detect a predominant replication defect for most of these viral mutants, concerning parameters of replication kinetics (qPCR), viral protein production (Western blot/CoIP) and capsid egress (confocal imaging/EM). Specifically, only the ORF-UL50 deletion rescue virus showed a block of genome synthesis during late stages of infection, whereas all phosphosite mutants exhibited marginal differences compared to wild-type or revertants. These results (i) emphasize a rate-limiting function of pUL50 for nuclear egress, and (ii) demonstrate that mutations in all mapped pUL50 phosphosites may be largely compensated. A refined mechanistic concept points to a multifaceted nuclear egress regulation, for which the dependence on the expression and phosphorylation of pUL50 is discussed.