Cellular restriction factors (RFs) act as important constitutive innate immune barriers against viruses. In 2006, the promyelocytic leukemia protein was described as the first RF against human cytomegalovirus (HCMV) infection which is antagonized by the viral immediate early protein IE1. Since then, at least 15 additional RFs against HCMV have been identified, including the chromatin regulatory protein SPOC1, the cytidine deaminase APOBEC3A and the dNTP triphosphohydrolase SAMHD1. These RFs affect distinct steps of the viral replication cycle such as viral entry, gene expression, the synthesis of progeny DNA or egress. This review summarizes our current knowledge on intrinsic immune mechanisms restricting HCMV replication as well as on the viral strategies to counteract the inhibitory effects of RFs. Detailed knowledge on the interplay between host RFs and antagonizing viral factors will be fundamental to develop new approaches to combat HCMV infection.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants.

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.

Programmed necrosis is an integral part of intrinsic immunity, serving to combat invading pathogens and restricting viral dissemination. The orchestration of necroptosis relies on a precise interplay within the necrosome complex, which consists of RIPK1, RIPK3 and MLKL. Human cytomegalovirus (HCMV) has been found to counteract the execution of necroptosis during infection. In this study, we identify the immediate-early 1 (IE1) protein as a key antagonist of necroptosis during HCMV infection. Infection data obtained in a necroptosis-sensitive cell culture system revealed a robust regulation of post-translational modifications (PTMs) of the necrosome complex as well as the importance of IE1 expression for an effective counteraction of necroptosis. Interaction analyses unveiled an association of IE1 and RIPK3, which occurs in an RHIM-domain independent manner. We propose that this interaction manipulates the PTMs of RIPK3 by promoting its ubiquitination. Furthermore, IE1 was found to exert an indirect activity by modulating the levels of MLKL via antagonizing its interferon-mediated upregulation. Overall, we claim that IE1 performs a broad modulation of innate immune signaling to impede the execution of necroptotic cell death, thereby generating a favorable environment for efficient viral replication.

ISG20 is an interferon-regulated protein that exhibits RNase activity thereby inhibiting the replication of a broad spectrum of RNA viruses. By single cell RNA sequencing, we identified ISG20 as an antiviral factor for human cytomegalovirus (HCMV) as it was upregulated in a population of HCMV-resistant cells. In accordance with an antiviral role on herpesviruses, overexpression of ISG20 in primary human fibroblasts led to reduced HCMV and HSV-1 replication, while knockdown of ISG20 enhanced virus growth. In Western blot kinetics, we observed that inhibition of HCMV replication by ISG20 occurs at the early stage of infection which correlated with reduced amounts of viral early and late transcripts. However, neither the half-life of viral and cellular RNAs nor of viral DNA was decreased in ISG20-expressing cells, indicating that ISG20 does not exert its antiviral effect via a degradation of RNAs or DNA. Instead, RNA-seq analysis revealed an innate immune defense signature upon ISG20 expression that comprised the upregulation of a distinct set of interferon stimulated genes (ISGs), zinc finger protein genes (ZNFs) and of transposable elements (TEs). Our data indicate that this gene signature augments both IFN production and response of the host cell. Consistently, the JAK-STAT inhibitor ruxolitinib rescued HCMV gene expression in ISG20-expressing cells. We conclude that ISG20 induces a broad immune defense signature that serves to amplify the IFN-mediated host cell defense thus explaining its extended antiviral activity. The interferon-induced protein 20 (ISG20) is best known for its antiviral function on many RNA viruses. Since in vitro experiments detected that this protein is capable of directly degrading RNA, it is thought that this enzymatic function acts as the prevailing mechanism of inhibition. In contrast, knowledge concerning the role of ISG20 for DNA viruses is limited. Here, we identify the ISG20 gene within an antiviral signature of primary human fibroblast that are resistant to the herpesvirus human cytomegalovirus (HCMV). Further experiments demonstrated that ISG20 severely impairs the replication of both HCMV and herpes simplex virus type I, suggesting a broad antiviral activity on herpesviruses. Surprisingly, neither cellular nor viral RNAs nor viral DNA were found to be degraded upon expression of ISG20. Transcriptomic analyses revealed a distinct upregulation of type I interferon (IFN) response genes, zinc finger protein genes and endogenous retroelements indicating a profound effect of ISG20 on host cells. We show that depletion of ISG20 severely impairs the host cell’s response to IFN which is consistent with the finding that interference with IFN-signaling alleviates the antiviral effect of ISG20 overexpression. In summary, we propose a novel mechanism of ISG20 action based on the induction of an innate immune defense signature which serves to amplify the host cell’s IFN response.

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication.