Human cytomegalovirus (CMV) dysregulates neurodevelopmental pathways in cerebral organoids.

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels.

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, 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 RORß, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells.

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.

Combined Treatment with Host-Directed and Anticytomegaloviral Kinase Inhibitors: Mechanisms, Synergisms and Drug Resistance Barriers.

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.

Autofluorescent Artemisinin-Benzimidazole Hybrids via Organo-Click Reaction: Study of Antiviral Properties and Mode of Action in Living Cells.

Drug modification by a fluorescent label is a common tool for studying its mechanism of action with fluorescence microscopy techniques. However, the attachment of a fluorescent label can significantly alter the polarity, solubility, and biological activity of the investigated drug, and, as a result, the studied mechanism of action can be misrepresented. Therefore, developing efficient drugs, which are inherently fluorescent and can be tracked directly in the cell is highly favorable. Here an easy formation of fluorescent hybrid drugs is presented, generated by a combination of two readily available non-fluorescent pharmacophores via a non-cleavable linker using a Ramachary-Bressy-Wang organocatalyzed azide-carbonyl [3+2] cycloaddition (organo-click) reaction. All newly prepared fluorescent compounds showed strong anti-HCMV activity (EC50 down to 0.07±0.00 µM), thus presenting a very promising drug developmental basis compared to the approved drug ganciclovir (EC50 2.60±0.50 µM). Remarkably, in vitro fluorescent imaging investigation of new compounds revealed induced changes in mitochondrial structures, which is a phenotypical hallmark of antiviral activity. This approach opens up new vistas for the easy formation of potent fluorescent drugs from readily available non-fluorescent parent compounds and might facilitate insight into their mode of action in living cells, avoiding the requirement of linkage to external fluorescent markers.

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target.

Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.

An antiviral targeting strategy based on the inducible interference with cytomegalovirus nuclear egress complex.

The nucleocytoplasmic capsid egress of herpesviruses like the human cytomegalovirus (HCMV) is based on a uniquely regulated process. The core nuclear egress complex (NEC) of HCMV, represented by the pUL50-pUL53 heterodimer, is able to oligomerize and thus to build hexameric lattices. Recently, we and others validated the NEC as a novel target for antiviral strategies. So far, the experimental targeting approaches included the development of NEC-directed small molecules, cell-penetrating peptides and NEC-directed mutagenesis. Our postulate states that an interference with the hook-into-groove interaction of pUL50-pUL53 prevents NEC formation and strictly limits viral replication efficiency. Here, we provide an experimental proof-of-concept of the antiviral strategy: the inducible intracellular expression of a NLS-Hook-GFP construct exerted a pronounced level of antiviral activity. The data provide evidence for the following points: (i) generation of a primary fibroblast population with inducible NLS-Hook-GFP expression showed nuclear localization of the construct, (ii) interaction between NLS-Hook-GFP and the viral core NEC was found specific for cytomegaloviruses but not for other herpesviruses, (iii) construct overexpression exerted a strong antiviral activity against three strains of HCMV, (iv) confocal imaging demonstrated the interference with NEC nuclear rim formation in HCMV-infected cells, and (v) quantitative nuclear egress assay confirmed the block of viral nucleocytoplasmic transition and, consequently, an inhibitory effect onto viral cytoplasmic virion assembly complex (cVAC). Combined, data confirmed that the specific interference with protein-protein interaction of the HCMV core NEC represents an efficient antiviral targeting strategy.

'Shared-Hook' and 'Changed-Hook' Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis.

Herpesviruses replicate their genomes and assemble their capsids in the host cell nucleus. To progress towards morphogenesis in the cytoplasm, herpesviruses evolved the strategy of nuclear egress as a highly regulated process of nucleo-cytoplasmic capsid transition. The process is conserved among α-, ß- and γ-herpesviruses and involves the formation of a core and multicomponent nuclear egress complex (NEC). Core NEC is assembled by the interaction between the nucleoplasmic hook protein, i.e., pUL53 (human cytomegalovirus, HCMV), and the integral membrane-associated groove protein, i.e., pUL50. Our study aimed at the question of whether a panherpesviral NEC scaffold may enable hook-into-groove interaction across herpesviral subfamilies. For this purpose, NEC constructs were generated for members of all three subfamilies and analyzed for multi-ligand interaction using a yeast two-hybrid (Y2H) approach with randomized pUL53 mutagenesis libraries. The screening identified ten library clones displaying cross-viral shared hook-into-groove interaction. Interestingly, a slightly modified Y2H screening strategy provided thirteen further changed-hook pUL53 clones having lost parental pUL50 interaction but gained homolog interaction. In addition, we designed a sequence-predicted hybrid construct based on HCMV and Epstein-Barr virus (EBV) core NEC proteins and identified a cross-viral interaction phenotype. Confirmation was provided by applying protein-protein interaction analyses in human cells, such as coimmunoprecipitation settings, confocal nuclear rim colocalization assays, and HCMV ΔUL53 infection experiments with pUL53-complementing cells. Combined, the study provided the first examples of cross-viral NEC interaction patterns and revealed a higher yield of human cell-confirmed binding clones using a library exchange rate of 3.4 than 2.7. Thus, the study provides improved insights into herpesviral NEC protein binding specificities of core NEC formation. This novel information might be exploited to gain a potential target scaffold for the development of broadly acting NEC-directed inhibitory small molecules.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

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.

Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses.

The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. One major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication, and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, a well-established substrate of pUL97 was no longer hyperphosphorylated. This effect was detectable as early as 4 h post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.

A Peptide Inhibitor of the Human Cytomegalovirus Core Nuclear Egress Complex.

The replication of human cytomegalovirus (HCMV) involves a process termed nuclear egress, which enables translocation of newly formed viral capsids from the nucleus into the cytoplasm. The HCMV core nuclear egress complex (core NEC), a heterodimer of viral proteins pUL50 and pUL53, is therefore considered a promising target for new antiviral drugs. We have recently shown that a 29-mer peptide presenting an N-terminal alpha-helical hook-like segment of pUL53, through which pUL53 interacts with pUL50, binds to pUL50 with high affinity, and inhibits the pUL50-pUL53 interaction in vitro. Here, we show that this peptide is also able to interfere with HCMV infection of cells, as well as with core NEC formation in HCMV-infected cells. As the target of the peptide, i.e., the pUL50-pUL53 interaction, is localized at the inner nuclear membrane of the cell, the peptide had to be equipped with translocation moieties that facilitate peptide uptake into the cell and the nucleus, respectively. For the resulting fusion peptide (NLS-CPP-Hook), specific cellular and nuclear uptake into HFF cells, as well as inhibition of infection with HCMV, could be demonstrated, further substantiating the HCMV core NEC as a potential antiviral target.

'Come together'-The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions.

Herpesviral nuclear egress is a fine-tuned regulatory process that defines the nucleocytoplasmic release of viral capsids. Nuclear capsids are unable to traverse via nuclear pores due to the fact of their large size; therefore, herpesviruses evolved to develop a vesicular transport pathway mediating the transition across the two leaflets of the nuclear membrane. The entire process involves a number of regulatory proteins, which support the local distortion of the nuclear envelope. In the case of the prototype species of ß-Herpesvirinae, the human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the core proteins pUL50 and pUL53 that oligomerize, form capsid docking lattices and mediate multicomponent assembly with NEC-associated viral and cellular proteins. The NEC-binding principle is based on the hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. Thus far, the function and characteristics of herpesviral core NECs have been well studied and point to the groove proteins, such as pUL50, as the multi-interacting, major determinants of NEC formation and egress. This review provides closer insight into (i) sequence and structure conservation of herpesviral core NEC proteins, (ii) experimentation on cross-viral core NEC interactions, (iii) the essential functional roles of hook and groove proteins for viral replication, (iv) an establishment of assay systems for NEC-directed antiviral research and (v) the validation of NEC as putative antiviral drug targets. Finally, this article provides new insights into the conservation, function and antiviral targeting of herpesviral core NEC proteins and, into the complex regulatory role of hook and groove proteins during the assembly, egress and maturation of infectious virus.

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors.

The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.

Synthesis, molecular features and biological activities of modified plant polysaccharides.

Plant polysaccharides represent a natural source material characterized by high ubiquity, abundance and biological activities that can be utilised as organic matter in numerous manufacturing processes including pharmaceutical. The goal-oriented modification of polysaccharides by chemical reactions brings about a substantial refinement in their molecular features and biological activity. An improved understanding of the intrinsic molecular features of plant polysaccharides, as well as the structure-activity relationship eventually caused by chemical alterations, may be fundamental for the development of technologies supporting their biomedical oriented exploitation. Herein, we attempt to provide a broader view on these topics and to illustrate methodologies of chemical modification. These are intended to enhance the functionality of plant polysaccharides, such as their modification-induced biological activities, finally making the products available for translational purposes.

Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations.

Human cytomegalovirus (HCMV) is a pathogenic human herpesvirus associated with serious, potentially life-threatening symptoms in the immunocompromised or immunonaïve host. The limitations encountered by antiviral therapy options currently available include a narrow panel of accessible targets, the induction of viral drug resistance as well as severe drug dosage-mediated side-effects. Improved drug-targeting strategies to resolve these issues are the focus of our investigations. In particular, pharmaceutical kinase inhibitors (PKIs), either directed to host kinases or directed to the viral protein kinase pUL97, have been considered to overcome these restrictions. Recently, we reported the identification of a synergistic combination of two PKIs directed to host cyclin-dependent kinase 7 (CDK7) and viral CDK ortholog pUL97. Here, we substantiate these findings with the following results: (i) true drug synergy was exhibited by various chemical classes of PKI pairs directed to pUL97 and CDK7; (ii) no putative amplification of cytotoxicity by these drug combinations was observed; (iii) a reduction in drug dosage levels for synergistic combinations was defined on a quantitative basis and compared to monotreatments; (iv) the quantities of target proteins CDK7 and pUL97 expressed in HCMV-infected cells were assessed by confocal imaging, indicating a strong down-modulation of CDK7 levels as a result of synergistic drug treatment; (v) the functional importance of these target kinases, both binding to cyclin H, was illustrated by assessing HCMV replication under the viral genomic deletion of ORF-UL97 or cellular cyclin knock-out; (vi) new combinations of HCMV-specific drug synergy were demonstrated for solely host-directed treatments using PKIs against CDK2, CDK7, CDK8 and/or CDK9 and (vii) a triple PKI combination provided further support for the synergy approach. With these combined findings, this study highlights the potential of therapeutic drug combinations of approved, developmental and preclinical PKIs for expanding future options for anti-HCMV therapy.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity.

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

Anti-SARS-CoV-2 Inhibitory Profile of New Quinoline Compounds in Cell Culture-Based Infection Models.

Invited for the cover of this issue are Manfred Marschall, Svetlana B. Tsogoeva and co-workers at Friedrich-Alexander University of Erlangen-Nürnberg. The image depicts a new anti-SARS-CoV-2 compound in front of SARS-CoV-2 viruses. Read the full text of the article at 10.1002/chem.202103861.