Regulatory protein kinases of herpesvirus infections - replication, pathogenesis and antiviral therapy
Human cytomegalovirus (HCMV) is a major opportunistic pathogen of humans with a worldwide distribution. Primary infection with HCMV in persons with a normal immune system is generally mild or asymptomatic, while in immunocompromised persons and in the situation of congenital infection, HCMV frequently causes systemic disease with severe clinical consequences. Most of the currently available anti-HCMV drugs face limitations in terms of unwarranted side-effects and the induction of drug resistance. Thus, the development of novel anti-HCMV drugs, and similarly also broader acting antiherpesviral drugs, is urgently required. For this, protein kinase inhibitors proved to be highly interesting drug candidates, as both cellular and herpesviral protein kinases represent important determinants of viral replication and pathogenicity.
The research concept of the group is focused on the following topics:
- Functional and structural characterization of the protein kinase pUL97 of HCMV
- Cross-talk between herpesviral and cellular kinases
- interactive regulation of the nuclear egress of HCMV and other herpesviruses
- modulation of cell cycle proteins by pUL97 during viral pathogenesis
- Kinase inhibitors as a new type of antiviral drugs.
The summarizing scheme illustrates the binding and phosphorylation of cellular (green) and viral (orange) substrate proteins by pUL97. For details, see Steingruber & Marschall, 2020, PMID: 32260430.
Functional analysis of the cytomegaloviral protein kinase pUL97
Herpesviral protein kinases are important determinants of the efficiency of viral replication. They fulfill a number of regulatory functions by phosphorylating both viral and cellular proteins. pUL97 of HCMV is the prototype of the UL-subfamily of serine/threonine-specific herpesviral protein kinases (HvUL kinases). An analysis of the structure-activity relationship of pUL97 and homologous kinases of other herpesviruses resulted in the definition of the kinase domain, the mapping of functionally important regions and the identification of phosphorylated substrates. Experimental settings to reveal the 3D structure of pUL97 are presently performed, including both approaches of structural biology and bioinformatics. As an important functional feature of pUL97, we demonstrated that this kinase is expressed in three isoforms arising from alternative translational initiation. Catalytic activity, including autophosphorylation and substrate phosphorylation, proved to be indistinguishable for two of the isoforms (M1 and M74), while its third isoform (M157) comprised functional differences. In particular, the interaction and phosphorylation of viral substrate proteins was less efficiently performed by the smallest isoform M157. These findings could be substantiated by the analysis of recombinant HCMVs expressing individual pUL97 isoforms. On this basis, also the question of pUL97-phosphorylated substrate proteins has been addressed by various experimental approaches. Currently, the picture became progressively clearer that the viral kinase pUL97 is a multifunctional regulator that binds and phosphorylates a number of cellular (Fig. 1, green) and viral (orange) substrate proteins.
For details, see Marschall et al., 2017 and 2020 (reviews); Milbradt et al., 2010, 2016 and 2018; Marschall et al., 2005; Sonntag et al., 2017; Kicuntod et al., 2021, PMID: 33799898; Häge et al., 2020, PMID: 32168891, and 2021, PMID: 33499341.
The regulatory importance of protein kinases for cytomegalovirus nuclear egress
The role of pUL97 within the HCMV replication cycle is manifested on 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 were able to demonstrate an involvement of pUL97 in the phosphorylation of nuclear lamins. The nuclear lamina is a rigid proteinaceous meshwork underlining the inner nuclear membrane. During infection with herpesviruses, the nuclear lamina restricts the efficiency of nucleocytoplasmic transport 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 lamin B receptor. pUL97 is able to phosphorylate lamins in a site-specific manner (Ser22) and 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 and pUL53. The pUL50-pUL53 interactor pair is considered as the core of the HCMV-specific nuclear egress complex (NEC) that contains a number of additional viral and cellular proteins. 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. This activity is highly suggestive to represent 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 (Fig. 2).
The secondary structure elements of the hook-like N-terminal extension of pUL53 (blue) are embracing pUL50 (red) and become buried in the interface formed by three pUL50 alpha-helices, thereby promoting the hook-into-groove interaction that is shown in a complex of hexameric arrangement. For details, see Walzer et al., 2015, PMID: 26432641; Muller et al., 2020, PMID: 31980459; Schweininger et al., 2021, JBC revision.
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 Å2 to the total interface area (hook-into-the-groove interaction; Fig. 3). The pUL50 structure reveals a considerable repositioning of its alpha-C helix upon pUL53 binding. 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.
Our biochemical and proteomic investigations of binding partners of the pUL50-pUL53 core complex suggested a combination of viral and cellular constituents associated within the multicomponent NEC in its final composition at late times of HCMV replication. In particular for pUL50, five binding partners could be identified, i.e. pUL53, PKC-alpha, 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, PKC-alpha and CDK1). These are currently investigated by mutagenesis and the transfer of mutant versions of ORF-UL50 into recombinant HCMVs. The ongoing functional investigation of the HCMV-specific NEC may reveal novel insights into these pronounced functional ties of virus-host interaction. In addition, the crystal structures of the EBV- and VZV-specific core NECs have been reported by our group recently, so that structural data are now available for alpha-, beta- and gamma-herpesviral NECs. This situation allowed for intensified in silico docking and biochemical inhibitor binding studies, with the aim to utilize the NEC as a novel target for the development of antiherpesviral drugs. As a first level of success, we could identify the inhibitory small molecule merbromin as a prototype blocker of pUL50–pUL53 interaction, thus exerting a proven anti-HCMV activity in cell culture-based models.
Cytomegalovirus-specific cellular signaling
It is an established concept that cytomegalovirus infection (or similarly infections with many other human herpesviruses) induce a cellular program of signaling that is characterized by the induced expression and activity of a variety of cellular protein kinases. This kind of upregulated signaling program provides a finger print, individually reflecting the parameters of replication and pathogenesis of a single virus (virus-specific cellular signaling, VSS). In case of HCMV, we established a novel approach of kinome profiling that suggests an important role of specific cellular protein kinases for viral replication. Experimental inhibition of such kinases showed a significant reduction, whereas inhibition of other kinases led to an activation of HCMV replication. Furthermore, analysis of the mode of action of those kinase inhibitors exerting antiviral activity (VSS inhibitors) suggested a substantial benefit for the efficiency of viral replication at the immediate early or early-late phases of HCMV gene expression. Interestingly, an ongoing comparative analysis of kinase patterns induced by various herpesviruses (as representatives of the alpha-, beta- and gamma-herpesvirus subfamilies) indicated partly conserved events of kinase/signaling upregulation and, for other kinases, virus-specific differences. Thus, our combined data provide new information on host cell kinases involved in viral replication and uncovered potential targets for future diagnostic or antiviral proceedings. The latter aspect is also addressed by our long-term analysis of the antiviral mode of activity of the broad antiinfective drug artesunate. Recent findings demonstrated an important point of the antiviral activity of artesunate in that it interferes with NF-kB -specific signaling. A direct binding of artesunate to NF-kB p65 is strongly suggested by our data. Notably, the enhanced anti-HCMV activity of synthetic artesunate dimers, trimers and hybrid molecules (cooperation with the group of Svetlana B. Tsogoeva) currently opens promising novel perspectives, not only in antiviral research, but also in the mechanistic analysis of HCMV specific cellular signaling. A mass spectrometry-based proteomic approach, using linker-coupled versions of artesunate compounds (Fig. 4A), revealed a selection of highly interesting drug target proteins, in particular including mitochondrial regulators. Importantly, the antiviral efficacy of artesunate compounds increased with the degree of multimerization (Fig. 4B; trimers>dimers>monomers). The functional validation of these proteins is currently performed, specifically taking into account a postulated regulatory link between mitochondrial activity and the efficiency of HCMV replication.
The model suggests a quaternary complex consisting of two pUL97 molecules (red, orange) bridged by two cyclin T1 domains (gray). pUL97(231–280) was modeled in an extended conformation in interface 2, whereas the globular pUL97 kinase domain was modeled to interface 1. Red and orange lines denote parts of the pUL97 structure for which no structural information is available. For details, see Schütz et al., 2021, PMID: 34198986.
Cyclins and cyclin-dependent protein kinases (CDKs) serve as coregulators of cytomegalovirus replication
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. For 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. Recently, we identified additional interactions between pUL97 and human cylins of types T1, B1 and H. As a specifically important aspect, the demonstration of pUL97-cyclin complexes by biochemical settings (CoIP), mass spectrometry-based proteomics and bioinformatic modeling addressed novel questions of the regulatory consequences cyclin-associated viral kinase pUL97. Our current 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. Very recently, we demonstrated the functional importance of the interaction between pUL97 and human cyclin T1 by the characterization of recombinant HCMVs that carry small deletions in pUL97 abrogating this interaction. On the basis of these data, the cyclin T1 binding region of pUL97, 231–280, proved to be an important functional determinant of pUL97 kinase activity and viral replicative fitness. Also, a molecular model of quaternary pUL97–cyclin T1 interaction could be established (Fig. 5).
In addition to the viral CDK ortholog pUL97, also cellular 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 data prove the high antiviral efficacy of inhibitors of virus-relevant CDKs. In particular, two highly potent and selective inhibitors of CDK7 exert a broad and effective antiviral activity. These findings may be highly beneficial for the development of novel broad-spectrum antiherpesviral drugs.
Experimental development of antiherpesviral drug candidates based on experimental protein kinase inhibitors
Cytomegaloviral pUL97 is considered as a validated target enzyme for novel antiviral drugs. This seems particularly interesting due to the fact that pUL97 is already an essential part of conventional antiviral therapy, carrying out a pacemaker phosphorylation reaction on ganciclovir and related nucleoside analogs. For the development of a pUL97-directed antiviral strategy, we established quantitation systems for pUL97 kinase activity and thereby identified novel chemical classes of compounds (quinazolines, indolocarbazoles, benzimidazoles and others) that inhibit the pUL97 kinase activity. Hereby, it was shown that these kinase inhibitors are able to 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 very recent 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 bioinformatics-, 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 identified shown for the CDK7 inhibitor LDC4297. This interesting compound, comprising an outstanding selectivity for CDK7, has been characterized by a kinome-wide evaluation (>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) and exerts a broad antiherpesviral activity (whereas non-herpesviruses, i.e. adeno-, pox-, retro- and orthomyxoviruses, showed only intermediate or no sensitivity). Recently, we reported an in vivo analysis, using MCMV infection of Rag-/- immunodeficient mice, thus providing a proof-of-concept for the antiviral potency of LDC4297. Thus, the CDK7 inhibitor LDC4297 is a highly potent tool for the analyses of mutual interregulation between HCMV and CDK and represents a candidate compound for the study of broadly acting antiherpesviral compounds. Very recently, we demonstrated the true synergistic potential of combination treatments using two kinase inhibitors directed to CDK7 and pUL97 (Fig. 6), a finding which is presently substantiated by a broader analysis of CDK-/pUL97-specific candidate drugs both in vitro and in vivo.
Analysis was performed with human Caco-2 cells using the indicated multiple readout system. Here, as an example, the antiviral activity of the viral 3CLpro protease inhibitor GC376 was assessed by a yellow fluorescence protein reporter measurement (YFP), a PCR determination of viral genomes in the cell culture supernatants (RT-qPCR), a fluorescence antibody-mediated detection of viral surface protein (mAb-S), and a Neutral Red-based measurement of drug cytotoxicity levels (NRA). For details, see Hahn et al., 2021, doi.org/10.3390/pathogens10091076.
Current issue of COVID-19: novel strategies to develop antiviral compounds on a broader basis, particularly focusing on anti-SARS-CoV-2 candidate drugs
The ongoing worldwide crisis initiated by the SARS-CoV-2 pandemic prompted us to extend our attempts of antiviral drug research also into this field. This analysis, focused on the search for potent small molecules directed against SARS-CoV-2, includes host-directed antivirals (HDAs) and direct-acting antivirals (DAAs) as well as combinatorial treatments with both. To this end, a methodological platform has been established, thus providing novel options of reporter-based detection of SARS-CoV-2 and a newly developed multi-readout assay system (MRA) to evaluate SARS-CoV-2 replication in cell culture models. This enables the quantitative assessment of compounds exerting anti-SARS-CoV-2 activity and thereby yielded first candidate drugs, such as IMU-838, GC376 and SNS032 that show promising antiviral efficacies in these systems (Hahn et al., 2020 and 2021).