Research Group of M. Marschall

The role of pUL97 within the HCMV replication cycle is manifested in several different stages, including viral DNA synthesis, phosphorylation-mediated inactivation of the cell cycle regulator Rb and nuclear viral capsid egress. Concerning the nuclear capsid egress, we demonstrated an involvement of pUL97 in the phosphorylation of nuclear lamins. The nuclear lamina is a rigid proteinaceous meshwork underlining the inner nuclear membrane. During herpesviral infections, the nuclear lamina restricts the efficiency of the nucleocytoplasmic transport of viral capsids, because of the large size of herpesviral capsids, which does not allow a direct transition through the nuclear pore complex. Lamina destabilization requires site-specific phosphorylation of lamins and lamin-binding membrane proteins. In case of HCMV, we demonstrated that pUL97 is recruited to the nuclear lamina through the interaction with a cellular multi-ligand binding protein, p32/gC1qR, which is also associated with the nuclear transmembrane protein emerin. pUL97 subsequently phosphorylates lamins of type A/C in a site-specific manner (Ser22) to induce a morphological alteration of the nuclear lamina. Thus, pUL97 has a crucial effect on the destabilization of the lamina, which is a prerequisite for the viral nuclear egress. In addition to pUL97, two other viral proteins are strictly associated with the nuclear lamina, namely pUL50 (groove protein) and pUL53 (hook protein). The pUL50–pUL53 interactor pair is considered as core of the HCMV-specific nuclear egress complex (NEC) that recruits a number of additional viral and cellular proteins towards a multicomponent complex. Importantly, the Ser22-specific lamin phosphorylation generates a binding motif for the cellular prolin-specific isomerase Pin1 that induces a cis/trans isomerization of Pro23 in lamin A/C. According to the present state of the art, the Pin1 activity represents the main mechanism of the herpesvirus-induced structural conversion of the nuclear lamina, thus leading to local lamina disassembly as a prerequisite for viral nuclear capsid egress (Figure 1).

Moreover, we were able to characterize the pUL50–pUL53 nuclear import and heterodimerization at the inner nuclear membrane. While pUL50 is a trans-membrane protein, directly inserted into the inner nuclear membrane, pUL53 is translocated to the nucleus via its recently mapped N-terminal NLS and is recruited to the inner nuclear rim by binding to pUL50. Thus, direct pUL50–pUL53 interaction is a prerequisite for intracellular trafficking and the fine-localization of both proteins. A hallmark of these investigations was the successful crystallization and resolution of the 3D structure of the globular domains of the pUL50–pUL53 core NEC of HCMV and other herpesviruses by our group and other investigators. The pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a crucial element of the pUL50–pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Ų to the total interface area (hook-into-groove interaction; Figure 2). The pUL50 structure reveals a considerable repositioning of its aC helix upon pUL53 binding. The resolution of additional herpesviral NECs, namely EBV (Muller et al., 2020, JBC) and VZV (Schweininger et al., 2022, JBC) by our group confirmed this unique NEC property, termed as herpesviral hook-into-groove interaction. A close examination of the crystal structure indicated partial assembly of pUL50–pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for the multimeric NEC. Moreover, our biochemical and proteomic investigations of binding partners of the pUL50–pUL53 complex suggested a combination of viral and cellular constituents associated with the multimeric NEC in its final composition at late stages of HCMV replication. In particular for pUL50, five binding partners were identified, i.e. pUL53, PKCa, p32/gC1qR, emerin and CDK1. A phospho-specific mass spectrometry analysis led to the identification of 14 major and minor sites of pUL50 phosphorylation (as putative sites for the pUL50-phosphorylating kinases pUL97, PKCa and CDK1).

The ongoing functional investigation of the HCMV-specific NEC may reveal novel insights into these pronounced functional ties of virus-host interaction. As far as current options of NEC-specific antiviral targeting are concerned, we see a number of highly relevant aspects: (i) the blocking of core NEC formation, (ii) the interference with activities of the multicomponent NEC (such as site-specific phosphorylation or inhibition of lamin-directed prolyl cis/trans isomerization), (iii) the interference with capsid-docking to the NEC, or (iv) the blocking of membrane specific activities during primary envelopment. Regarding the situation of a multifactorial regulation of the herpesviral nuclear egress-specific mechanism, additional strategies of NEC interference appear possible. It should be emphasized that we already identified the first prototypes of NEC-inhibitory small molecules, such as merbromin (MBM; Figure 3), ibrutinib (IBM), LDC599, and others. Along with the characterization of these new core NEC inhibitors, we recognized that the property of covalent target binding of compounds proved to be highly advantageous for the efficacy of PPI inhibition. Therefore, the study of these covalent binders, termed 'warheads', recently attracted our closer interest. The mechanistic analyses of warheads, either directed against NEC, kinases or other regulatory viral proteins are presently ongoing. Moreover, an indirect NEC-associated prototype inhibitor was characterized with maribavir (MBV). This drug is directed to the viral kinase pUL97, which is a crucial codeterminant of NEC regulation, so that MBV exerts a strong nuclear egress-inhibitory activity. MBV has recently been clinically approved as the only kinase inhibitor within the entire field of antiviral therapy.

An important focus of our investigations is the identification of central phosphorylation pathways of cellular protein kinases which are essential for the replication of herpesviruses. In this regard, the phosphorylation of viral regulator proteins by cellular protein kinases is particularly interesting. In the case of HCMV, the specific interaction between viral proteins and cellular protein kinases, especially cyclin-dependent protein kinases (CDKs), is presently investigated. The identification of regulatory consequences of such interactions will define novel aspects of virus-host cell interaction. Our recent data confirmed previous reports that CDK activity modulates the intracellular localization of the HCMV nuclear regulatory protein pUL69. CDK inhibitors induce a nuclear speckled aggregation of pUL69 in HCMV-infected fibroblasts. Moreover, experimental data proved a direct protein interaction between pUL69 and CDK9/cyclin T. Interestingly, we identified that the phosphorylation of pUL69 can be mediated through cellular CDK as well as viral pUL97 kinase activities. This finding supported the current postulate that pUL97 represents a viral CDK ortholog. We identified additional interactions between pUL97 and human cyclins of types T1, B1 and H (Figure 4).

As a specifically important aspect, the demonstration of pUL97-cyclin complexes by biochemical settings (CoIP), mass spectrometry-based proteomics and bioinformatic modeling (Figure 5) addressed novel questions of regulatory consequences of the cyclin-associated viral kinase pUL97. Our concept suggests that the cyclins bound to pUL97 may recruit substrate proteins and may reinforce preferential events of pUL97-mediated substrate phosphorylation. Mutational analysis is presently performed and a CoIP-based interactomic analysis of pUL97 site-directed replacement and deletion mutants carrying putative defects in cyclin interaction refined our insight into the underlying regulatory details. Specific findings of the ongoing investigations are: (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 kinase activity and phosphorylation or HCMV-specific cyclin modulation; (ii) pUL97-mediated in vitro phosphorylation of cyclins is measurable for type B1 but not H, and no evidence is provided for mutual transphosphorylation between pUL97 and CDK7; and (iii) a cyclin T1/H-mediated bridging mechanism of pUL97 self-interaction is supported by current data. Concerning our approaches to define the functional relevance and molecular mode of interaction between pUL97 and human cyclins, we recently identified the following: (i) vCDK/pUL97–cyclin interaction was found conserved in all analyzed HCMV mutants, and viral replication fitness was almost indistinguishable from wildtype; (ii) deletion mutagenesis helped to map the interface 2 (IF2), the deletion of which resulted in a loss of interaction with cyclins T1 and H (but not B1), as well as a severely impaired viral replication efficiency; (iii) upon IF2 deletion, pUL97 kinase activity was significantly reduced, thus proving the functional importance of the pUL97–cyclin interaction; (iv) knock-out and knock-down experiments confirmed the importance of cyclin H as a major determinant of HCMV replication efficiency; (v) a quantitative in vitro kinase assay (qSox-IVKA) showed a significant increase of pUL97 kinase activity proportionally with the presence of cyclin H; and (vi) biochemical and bioinformatic analyses demonstrated a ternary complex vCDK/pUL97–cyclin H–CDK7 (Figure 5), which (vii) enables a transstimulation of CDK7 through pUL97 (but not vice versa).

As a consequence of these investigations, a first covalently binding compound that inhibited the pUL97–cyclin H interaction was identified with potent anti-HCMV activity, pointing towards another novel antiviral targeting strategy (Figure 6).

In addition, even more host CDKs have been characterized as coregulators of herpesviral replication by studies of our group and further investigators. Currently, we are performing a characterization of the interaction patterns and regulatory roles of individual CDKs (most of all CDK7) during HCMV infection of human fibroblasts and other cell types. Especially, CDKs and further protein kinases that possess importance for the replication of HCMV as well as other herpesviruses have been considered as potential antiviral drug candidates. In this regard, it appears promising that our current in vitro and also first in vivo data have proven the high antiviral efficacy of inhibitors of virus-relevant CDKs. In particular, a highly potent and selective inhibitor of CDK7, LDC4297, exerts a broad and effective antiviral activity. Interestingly, quantitative approaches of combination treatments identified statistically significant drug synergy levels between inhibitors of CDKs (LDC4297 and others) and vCDK/pUL97 (MBV and others). A mechanistic investigation of the synergy between these drugs pointed to specific advantages of this combination strategy (Figure 7), namely (i) a reduction of effective drug dosages, (ii) a novel kinase-based antiviral mode of action, and (iii) an improvement in drug resistance barriers. According to our current state of investigations, we are describing the further development of enhanced derivatives of parental CDK7 inhibitor, LDC4297, namely QRS compounds with the ability to covalently bind to this host target. Through this modification, an enhancement of anti-HCMV efficacy in vitro has been improved into the picomolar range of drug concentrations. Combined, these findings may be highly beneficial for the development of novel broad-spectrum antiherpesviral drugs.

Considering the current repertoire of approved antiherpesviral drugs, it appears striking that the vast majority is provided by direct-acting antivirals (DAAs). This may have practical reasons in the history of antiviral drug development, since the strategy was originally derived from the early recognition of virus-encoded targets and target structures that are unique or differ from their host orthologs. DAAs have been proven to be clinically highly expedient, their achievements and benefits in treatment, however, can be strongly limited. In particular with long-term therapeutic or prophylactic treatments, the induction of viral drug resistance, or unwarranted side-effects. For these reasons, an additional characterization of host-directed antivirals (HDAs) is presently aspired. Host-directed drugs, in particular when recognizing pathogenesis-related mutants of target proteins, have been greatly successful in areas spanning the treatments of tumor, metabolic, infectious, and inflammatory diseases. However, within the field of antiviral therapy, this topic misses in-depth experience, since only quite few HDAs have been clinically approved and utilized in therapeutic settings thus far.

Considering anti-HCMV drug targeting options, both DAAs and HDAs have been analyzed in more detail during the last decades (Figure 8). Hereby the cytomegaloviral kinase pUL97 has been validated as a target enzyme for novel antiviral drugs, with maribavir (MBV) as the first kinase-directed DAA that has recently been approved. This targeting strategy seems particularly interesting due to the fact that pUL97 has already been an essential part of conventional antiviral therapy for a long time, as it carries out a pacemaker phosphorylation reaction on ganciclovir and related nucleoside analogs. For developmental works of the pUL97-directed antiviral strategy in our own group, quantitation systems for pUL97 kinase activity have been established, thereby leading to the identification of novel chemical classes of compounds (quinazolines, indolocarbazoles, benzimi-dazoles, others) that inhibit the pUL97 kinase activity. Hereby, it was shown that these kinase inhibitors block HCMV replication efficiently in vitro and in vivo and that the inhibitory effects on the levels of kinase activity and virus replication are linked. Our study of quinazoline-type inhibitors of pUL97 indicated the high value of bioinformatic-based modeling approaches in the optimization of candidate compounds. An enlarged platform combining biochemistry-, bioinformatic-, structural biology- and medicinal chemistry-based approaches may substantially take forward our efforts of pUL97-directed drug design.

In addition to the analysis of pUL97 inhibitors, a broad-spectrum antiherpesviral activity was also identified for the CDK7 inhibitor LDC4297. This interesting compound comprises an outstanding selectivity for CDK7 (as characterized by a kinome-wide evaluation of >330 kinases profiled in vitro). LDC4297 exerts an efficient inhibition of HCMV replication in primary human fibroblasts at nanomolar concentrations (EC₅₀ 24.5±1.3 nM), proved efficacy also in vivo in a MCMV/mouse model, and moreover, provides a broad antiherpesviral activity. In comparison, non-herpesviruses, i.e. adeno-, pox-, retro- and orthomyxoviruses, showed either intermediate or no sensitivity. These results provided a proof-of-concept for the antiviral potency of the targeting of host CDK7 by LDC4297 or even enhanced-efficacy derivatives.

On an even broader scale, we demonstrated the importance of additional host CDKs for HCMV replication, including CDK2, CDK4/6, CDK9 and, very recently, CDK8. In particular, CDK8 ranged into the focus of our specific interest and might represent another highly interesting drug target. This is particularly true when considering the modern options for chemical modification of CDK target binders, i.e. through the coupling of a degrader unit. This strategy is based on the use of 'Proteolysis Targeting Chimeras' (PROTACs), which are hetero-bifunctional molecules containing two small molecule-binding ligands joined together by a linker. Such chemical linkage of PROTAC moieties is a means of recruiting a specific E3 ligase, thereby inducing the proteasome-mediated degradation of the target protein. Such PROTAC approaches, which are intended to enhance antiviral drug activity compared to their parental, non-degrading inhibitors, are especially worked out in the context of CDK targeting, so that the generation of an efficient CDK8-PROTAC is being intensely pursued. Thus, the longer-term objectives of this research line are the assessment of broadly acting antiherpesviral prototype compounds and the specific nomination of candidate drugs or combinatorial drug synergies. Beyond that, the use of CDK inhibitors in our projects provides a powerful tool for the analysis of mutual interregulation between HCMV and the host CDK–cyclin machinery.

Very similar approaches of drug targeting are now also developed for additional herpesviruses, such as Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), rhesus macaque cytomegalovirus (RhCMV), and for a panel of non-herpesviruses. For these virus systems, specific reporter modules have been established, which now allow for the analysis of regulatory details of virus replication in host cell types and for the assessment of antiviral candidate drugs.

Concerning our comparative analyses with non-herpesviruses, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORβ, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis (Figure 9). Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules. For future studies from 2024 on, this investigation of the RORγ role in virus infections will be further developed by Friedrich Hahn as an independent research line.