Differential upregulation of host cell protein kinases by the replication of α-, ß- and γ-herpesviruses provides a signature of virus-specific signalling.

Infections with human herpesviruses share several molecular characteristics, but the diversified medical outcomes are distinct to viral subfamilies and species. Notably, both clinical and molecular correlates of infection are a challenging field and distinct patterns of virus-host interaction have rarely been defined; this study therefore focuses on the search for virus-specific molecular indicators. As previous studies have demonstrated the impact of herpesvirus infections on changes in host signalling pathways, we illustrate virus-modulated expression levels of individual cellular protein kinases. Current data reveal (i) α-, ß- and γ-herpesvirus-specific patterns of kinase modulation as well as (ii) differential levels of up-/downregulated kinase expression and phosphorylation, which collectively suggest (iii) defined signalling patterns specific for the various viruses (VSS) that may prove useful for defining molecular indicators. Combined, the study confirms the correlation between herpesviral replication and modulation of signalling kinases, possibly exploitable for the in vitro characterization of viral infections.

Chemically sulfated polysaccharides from natural sources: Assessment of extraction-sulfation efficiencies, structural features and antiviral activities.

The provisioning of compound libraries with a high degree of diversity and attractive pharmacological properties is a limiting step in drug development. This study reports the production of highly bioactive sulfated polysaccharides, originally present in a nonsulfated, dormant state in natural sources, and demonstrates their antiviral activity (human cytomegalovirus EC50 values of 2.34-7.77 µg/mL) at a low degree of cytotoxicity. Furthermore, data strongly suggested the inhibition of virus entry as the main mode of antiviral action. Remarkably, the utilized oleum-DMF reagent was able to generate a range of sulfated polysaccharides from various natural sources, possessing varying saccharide compositions, degrees of sulfation (0.4-1.7) and molecular masses (38-94,000 g/mol). Typically, in a matter of minutes, this reagent not only solubilized polysaccharides but also chemically converted their hydroxyl functionality into sulfates. The most active sulfated polysaccharide (EC50 of 2.62 µg/mL) proved to be a 94,000 g/mol branched glucan with sulfates at C-6/C-3,6/C-2,3,6 positions. In conclusion, the important determinants of such compounds' antiviral activity are: (i) degree of sulfation, (ii) molecular mass and (iii) structural features. Thus, our approach offers a huge prospect for the improvement of natural source-derived libraries based on biologically active polysaccharides with diversified chemical profiles.

Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylation-regulated kinases during placental replication.

INTRODUCTION: Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication. METHODS: DYRK expression was investigated in AD169- and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR. RESULTS: HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3-fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p < 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p < 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p < 0.05) indicating these cellular kinases are required during HCMV placental replication. CONCLUSION: HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

Novel cytomegalovirus-inhibitory compounds of the class pyrrolopyridines show a complex pattern of target binding that suggests an unusual mechanism of antiviral activity.

Human cytomegalovirus (HCMV) is a major human pathogen with seropositivity rates in the adult population ranging between 40% and 95%. HCMV infection is associated with severe pathology, such as life-threatening courses of infection in immunocompromised individuals and neonates. Current standard therapy with valganciclovir has the disadvantage of adverse side effects and viral drug resistance. A novel anti-HCMV drug, letermovir, has been approved recently, so that improved therapy options are available. Nevertheless, even more so far unexploited classes of compounds and molecular modes of action will be required for a next generation of antiherpesviral treatment strategies. In this study, we focused on the analysis of the antiviral potency of a novel class of compounds, i.e. pyrrolopyridine analogs, and identified both hit compounds and their target protein candidates. In essence, we provide novel evidence as follows: (i) screening hit SC88941 is highly active in inhibiting HCMV replication in primary human fibroblasts with an EC50 value of 0.20 ±â€¯0.01 µM in the absence of cytotoxicity, (ii) inhibition occurs at the early-late stage of viral protein production and shows reinforcing effects upon LMV cotreatment, (iii) among the viruses analyzed, antiviral activity was most pronounced against ß-herpesviruses (HCMV, HHV-6A) and intermediate against adenovirus (HAdV-2), (iv) induction of SC88941 resistance was not detectable, thus differed from the induction of ganciclovir resistance, (v) a linker-coupled model compound was used for mass spectrometry-based target identification, thus yielding several drug-binding target proteins and (vi) a first confocal imaging approach used for addressing intracellular effects of SC88941 indicated qualitative and quantitative alteration of viral protein expression and localization. Thus, our findings suggest a multifaceted pattern of compound-target binding in connection with an unusual mode of action, opening up further opportunities of antiviral drug development.

Synthesis of Thymoquinone-Artemisinin Hybrids: New Potent Antileukemia, Antiviral, and Antimalarial Agents.

A series of hybrid compounds based on the natural products artemisinin and thymoquinone was synthesized and investigated for their biological activity against the malaria parasite Plasmodium falciparum 3D7 strain, human cytomegalovirus (HCMV), and two leukemia cell lines (drug-sensitive CCRF-CEM and multidrug-resistant subline CEM/ADR5000). An unprecedented one-pot method of selective formation of C-10α-acetate 14 starting from a 1:1 mixture of C-10α- to C-10ß-dihydroartemisinin was developed. The key step of this facile method is a mild decarboxylative activation of malonic acid mediated by DCC/DMAP. Ether-linked thymoquinone-artemisinin hybrids 6a/b stood out as the most active compounds in all categories, while showing no toxic side effects toward healthy human foreskin fibroblasts and thus being selective. They exhibited EC50 values of 0.2 µM against the doxorubicin-sensitive as well as the multidrug-resistant leukemia cells and therefore can be regarded as superior to doxorubicin. Moreover, they showed to be five times more active than the standard drug ganciclovir and nearly eight times more active than artesunic acid against HCMV. In addition, hybrids 6a/b possessed excellent antimalarial activity (EC50 = 5.9/3.7 nM), which was better than that of artesunic acid (EC50 = 8.2 nM) and chloroquine (EC50 = 9.8 nM). Overall, most of the presented thymoquinone-artemisinin-based hybrids exhibit an excellent and broad variety of biological activities (anticancer, antimalarial, and antiviral) combined with a low toxicity/high selectivity profile.

Deeper Insight into the Six-Step Domino Reaction of Aldehydes with Malononitrile and Evaluation of Antiviral and Antimalarial Activities of the Obtained Bicyclic Products.

The straightforward and efficient synthesis of complex aza- and carbobicyclic compounds, which are of importance for medicinal chemistry, is a challenge for modern chemical methodology. An unprecedented metal-free six-step domino reaction of aldehydes with malononitrile was presented in our previous study to provide, in a single operation, these bicyclic nitrogen-containing molecules. Presented here is a deeper investigation of this atom-economical domino process by extending the scope of aldehydes, performing post-modifications of domino products, applying bifunctional organocatalysts and comprehensive NMR studies of selected domino products. The thermodynamic aspects of the overall reaction are also demonstrated using DFT methods in conjunction with a semi-empirical treatment of van der Waals interactions. Furthermore, biological studies of seven highly functionalized and artemisinin-containing domino products against human cytomegalovirus (HCMV) and Plasmodium falciparum 3D7 are presented. Remarkably, in vitro tests against HCMV revealed five domino products to be highly active compounds (EC50 0.071-1.8 µm), outperforming the clinical reference drug ganciclovir (EC50 2.6 µm). Against P. falciparum 3D7, three of the investigated artemisinin-derived domino products (EC50 0.72-1.8 nm) were more potent than the clinical drug chloroquine (EC50 9.1 nm).

Facile access to potent antiviral quinazoline heterocycles with fluorescence properties via merging metal-free domino reactions.

Most of the known approved drugs comprise functionalized heterocyclic compounds as subunits. Among them, non-fluorescent quinazolines with four different substitution patterns are found in a variety of clinically used pharmaceuticals, while 4,5,7,8-substituted quinazolines and those displaying their own specific fluorescence, favourable for cellular uptake visualization, have not been described so far. Here we report the development of a one-pot synthetic strategy to access these 4,5,7,8-substituted quinazolines, which are fluorescent and feature strong antiviral properties (EC50 down to 0.6±0.1 µM) against human cytomegalovirus (HCMV). Merging multistep domino processes in one-pot under fully metal-free conditions leads to sustainable, maximum efficient and high-yielding organic synthesis. Furthermore, generation of artesunic acid-quinazoline hybrids and their application against HCMV (EC50 down to 0.1±0.0 µM) is demonstrated. Fluorescence of new antiviral hybrids and quinazolines has potential applications in molecular imaging in drug development and mechanistic studies, avoiding requirement of linkage to external fluorescent markers.

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 µM/SC84227, 0.60 ± 0.02 µM/SC97202, 6.26 ± 1.64 µM/SC97208, 0.71 ± 0.019 µM/Harmine and 0.63 ± 0.23 µM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

Synthesis of Novel Hybrids of Quinazoline and Artemisinin with High Activities against Plasmodium falciparum, Human Cytomegalovirus, and Leukemia Cells.

Many quinazoline derivatives have been synthesized over the last few decades with great pharmacological potential, such as antimalarial, anti-inflammatory, antimicrobial, anticancer, and antiviral. But so far, no quinazoline-artemisinin hybrids have been reported in the literature. In the present study, five novel quinazoline-artemisinin hybrids were synthesized and evaluated for their in vitro biological activity against malarial parasites (Plasmodium falciparum 3D7), leukemia cells (CCRF-CEM and CEM/ADR5000), and human cytomegalovirus. Remarkably, hybrid 9 (EC50 = 1.4 nM), the most active antimalarial compound of this study, was not only more potent than artesunic acid (EC50 = 9.7 nM) but at the same time more active than the clinically used drugs dihydroartemisinin (EC50 = 2.4 nM) and chloroquine (EC50 = 9.8 nM). Furthermore, hybrids 9 and 10 were the most potent compounds with regard to anticytomegaloviral activity (EC50 = 0.15-0.21 µM). They were able to outperform ganciclovir (EC50 = 2.6 µM), which is the relevant standard drug of antiviral therapy, by a factor of 12-17. Moreover, we identified a new highly active quinazoline derivative, compound 14, that is most effective in suppressing cytomegalovirus replication with an EC50 value in the nanomolar range (EC50 = 50 nM). In addition, hybrid 9 exhibited an antileukemia effect similar to that of artesunic acid, with EC50 values in the low micromolar range, and was 45 times more active toward the multidrug-resistant CEM/ADR5000 cells (EC50 = 0.5 µM) than the standard drug doxorubicin.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97.

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9-cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69-cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.

The broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa B (NF-κB) pathway by targeting RelA/p65.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of standard antiviral therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling inhibitors. The broad antiinfective drug artesunate (ART) offers additional therapeutic options such as oral bioavailability and low levels of toxic side-effects. Here, novel ART-derived compounds including dimers and trimers were synthesized showing further improvements over the parental drug. Antiviral activity and mechanistic aspects were determined leading to the following statements: (i) ART exerts antiviral activity towards human and animal herpesviruses, (ii) no induction of ART-resistant HCMV mutants occurred in vitro, (iii) chemically modified derivatives of ART showed strongly enhanced anti-HCMV efficacy, (iv) NF-κB reporter constructs, upregulated during HCMV replication, could be partially blocked by ART treatment, (v) ART activity analyzed in stable reporter cell clones indicated an inhibition of stimulated NF-κB but not CREB pathway, (vi) solid-phase immobilized ART was able to bind to NF-κB RelA/p65, and (vii) peptides within NF-κB RelA/p65 represent candidates of ART binding as analyzed by in silico docking and mass spectrometry. These novel findings open new prospects for the future medical use of ART and ART-related drug candidates.

The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

Highly potent artemisinin-derived dimers and trimers: Synthesis and evaluation of their antimalarial, antileukemia and antiviral activities.

New pharmaceutically active compounds can be obtained by modification of existing drugs to access more effective agents in the wake of drug resistance amongst others. To achieve this goal the concept of hybridization was established during the last decade. We employed this concept by coupling two artemisinin-derived precursors to obtain dimers or trimers with increased in vitro activity against Plasmodiumfalciparum 3D7 strain, leukemia cells (CCRF-CEM and multidrug-resistant subline CEM/ADR5000) and human cytomegalovirus (HCMV). Dimer 4 (IC50 of 2.6 nM) possess superior antimalarial activity compared with its parent compound artesunic acid(3) (IC50 of 9.0 nM). Dimer5 and trimers6 and 7 display superior potency against both leukemia cell lines (IC50 up to 0.002 µM for CCRF-CEM and IC50 up to 0.20 µM for CEM/ADR5000) and are even more active than clinically used doxorubicin (IC50 1.61 µM for CEM/ADR5000). With respect to anti-HCMV activity, trimer6 is the most efficient hybrid (IC50 0.04 µM) outperforming ganciclovir (IC50 2.6 µM), dihydroartemisinin(IC50 >10 µM) and artesunic acid (IC50 3.8 µM).

A novel CDK7 inhibitor of the Pyrazolotriazine class exerts broad-spectrum antiviral activity at nanomolar concentrations.

Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.

Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus.

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.

Using multi-channel level sets to measure the cytoplasmic localization of HCMV pUL97 in GFP-B-gal fusion constructs.

Human cytomegalovirus UL97-encoded protein kinase (pUL97) phosphorylates cellular and viral proteins and is critical for viral replication. To quantify the efficiency of nuclear translocation and to elucidate the role of putative nuclear localization signal (NLS) elements, immunofluorescence analysis of different pUL97 expression constructs was performed. Since manual quantitation of respective expression levels lacks objectivity and reproducibility, and is time-consuming as well, a computer-based model is established. This model enables objective quantitation of the degree of cytoplasmic localization λ. To determine the degree of cytoplasmic localization of different pUL97-GFP-ß-gal fusion proteins automatically, a multi-channel segmentation of the nucleus and cytoplasm of transfected HeLa cells is performed in DAPI and GFP micrographs. A watershed transform-based segmentation scheme is used for the segmentation of the cell nuclei. Subsequently, the cytoplasm is segmented using a fast marching level set method. Based on the segmentation of cell nuclei and cytoplasm, λ can be determined for each HeLa cell by quantitation of the ratio of average signal intensity outside and inside the nucleus. The degree of cytoplasmic localization of an individual construct is then determined by evaluating the average and standard deviation of λ for the corresponding HeLa cells. Evaluation demonstrates that nuclear transport of pUL97 is a multilayered mechanism resulting in different efficiencies of nuclear translocation between a small and a large isoform and objective quantitation of the cytoplasmic localization is possible with a high accuracy (96.7% and 94.3%).

Chemically engineered sulfated glucans from rice bran exert strong antiviral activity at the stage of viral entry.

Attachment and entry of many viruses are mediated by their affinity for polysaccharides present on the surface of target cells. In this paper, we demonstrate that sulfated glucans isolated from rice (Oryza sativa) can be utilized as experimental drugs exerting strong antiviral activity. In particular, oleum-DMF-based extraction is described as a procedure for the generation of chemically engineered glucans from commercially available rice bran. The one-step procedure has the potential to provide a spectrum of related glucans with varying molecular masses and modifications, including sulfation. The sulfated glucans P444, P445, and P446 possess increased antiviral activity compared to a previously described glucan (S1G). P444, P445, and P446 were highly active against human cytomegalovirus (HCMV), moderately active against other members of the family Herpesviridae, while not active against unrelated viruses. Specific experimentation with HCMV-infected cells provided evidence that antiviral activity was based on inhibition of viral entry and that inhibition occurred in the absence of drug-induced cytotoxicity. These findings underline the high potential of sulfated glucans for antiviral research and drug development. In addition, the procedure described for the efficient transformation of glucan hydroxy groups to sulfate groups may be similarly beneficial for the chemical alteration of other natural products.

Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis.

Currently available antiviral drugs frequently induce side-effects or selection of drug-resistant viruses. We describe a novel antiviral principle based on targeting the cellular enzyme dihydroorotate dehydrogenase (DHODH). In silico drug design and biochemical evaluation identified Compound 1 (Cmp1) as a selective inhibitor of human DHODH in vitro (IC50 1.5±0.2nM). Crystallization data specified the mode of drug-target interaction. Importantly, Cmp1 displayed a very potent antiviral activity that could be reversed by co-application of uridine or other pyrimidine precursors, underlining the postulated DHODH-directed mode of activity. Human and animal cytomegaloviruses as well as adenoviruses showed strong sensitivity towards Cmp1 in cell culture-based infection systems with IC50 values in the low micromolar to nanomolar range. Particularly, broad inhibitory activity was demonstrated for various types of laboratory and clinically relevant adenoviruses. For replication of human cytomegalovirus in primary fibroblasts, antiviral mode of activity was attributed to the early stage of gene expression. A mouse in vivo model proved reduced replication of murine cytomegalovirus in various organs upon Cmp1 treatment. These findings suggested Cmp1 as drug candidate and validated DHODH as a promising cellular target for antiviral therapy.