Broad-spectrum antiviral diazadispiroalkane core molecules block attachment and cell-to-cell spread of herpesviruses.

Regarding the problems with the current available drugs many research studies deal with the class of the dispirotripiperazine (DSTP)-based compounds. These are small molecules consisting of polycyclic saturated ring systems with positively charged nitrogen atoms. These compounds can interact with negatively charged HSPGs and thus block viral attachment. In a previous paper by Adfeldt et al. (2021), we have shown that the diazadispiroalkane derivatives 11826091 and 11826236 exhibit dose-dependent antiviral activity against human cytomegalovirus (HCMV) and pseudorabies virus (PrV). In the present study, these two small molecules are evaluated against two other herpesvirus species, murine cytomegalovirus (MCMV) and herpes simplex virus type 1 (HSV-1), as well as a HCMV clinical isolate. They exhibit potent antiherpetic activity against these herpesviruses with a high selectivity index. The low cytotoxicity was underlined by the LD50 determination in mice. We have shown that inhibition occurs at an early stage of infection. Interestingly, 11826091 and 11826236 reduced immediate early gene expression in HCMV and HSV-1 infected cells in a dose-dependent manner. Both small molecules probably interact electrostatically with sulfated glycosaminoglycans (GAGs) of proteoglycans on target cells resulting in blockage of adsorption sites for herpesvirus glycoprotein. Moreover, both compounds showed significant effects against the cell-associated viral spread of HSV-1 and HCMV. Overall, this study shows that 11826091 and 11826236 represent two promising candidates for a new approach of a broad antiviral therapy.

High-Sulfated Glycosaminoglycans Prevent Coronavirus Replication.

Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy.

Small Molecules-Prospective Novel HCMV Inhibitors.

Human cytomegalovirus (HCMV), a member of the betaherpesvirinae, can cause life-threatening diseases. HCMV is globally widespread, with a seroprevalence in adults varying from 50 to 100%. HCMV infection is rarely of significant consequence in immunocompetent individuals. However, although immune control is efficient, it cannot achieve the clearance of the virus. HCMV persists lifelong in the infected host and reactivates in certain circumstances. In neonates and in immunocompromised adults, HCMV is a serious pathogen that can cause fatal organ damage. Different antiviral compounds alone or in combination have been used for the treatment of HCMV diseases. In clinical use, mutations in the viral DNA polymerase or the terminase confer resistance to ganciclovir, foscarnet, cidofovir, and letermovir. There is an urgent need to find new well-tolerated compounds supporting different modes of action. The list of novel small molecules that might have anti-HCMV activity has grown in recent years. In this short review, a selection of compounds in clinical trials and novel inhibitors targeting host-cell factors or viral proteins is presented, and their modes of action, described.

Diazadispiroalkane Derivatives Are New Viral Entry Inhibitors.

Herpesviruses are widespread and can cause serious illness. Many currently available antiviral drugs have limited effects, result in rapid development of resistance, and often exhibit dose-dependent toxicity. Especially for human cytomegalovirus (HCMV), new well-tolerated compounds with novel mechanisms of action are urgently needed. In this study, we characterized the antiviral activity of two new diazadispiroalkane derivatives, 11826091 and 11826236. These two small molecules exhibited strong activity against low-passage-number HCMV. Pretreatment of cell-free virus with these compounds greatly reduced infection. Time-of-addition assays where 11826091 or 11826236 was added to cells before infection, before and during infection, or during or after infection demonstrated an inhibitory effect on early steps of infection. Interestingly, 11826236 had an effect by addition to cells after infection. Results from entry assays showed the major effect to be on attachment. Only 11826236 had a minimal effect on penetration comparable to heparin. Further, no effect on virus infection was found for cell lines with a defect in heparan sulfate expression or lacking all surface glycosaminoglycans, indicating that these small molecules bind to heparan sulfate on the cell surface. To test this further, we extended our analyses to pseudorabies virus (PrV), a member of the Alphaherpesvirinae, which is known to use cell surface heparan sulfate for initial attachment via nonessential glycoprotein C (gC). While infection with PrV wild type was strongly impaired by 11826091 or 11826236, as with heparin, a mutant lacking gC was unaffected by either treatment, demonstrating that primary attachment to heparan sulfate via gC is targeted by these small molecules.

HCMV-Mediated Interference of Bortezomib-Induced Apoptosis in Colon Carcinoma Cell Line Caco-2.

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

Dispirotripiperazine-core compounds, their biological activity with a focus on broad antiviral property, and perspectives in drug design (mini-review).

Viruses are obligate intracellular parasites and have evolved to enter the host cell. To gain access they come into contact with the host cell through an initial adhesion, and some viruses from different genus may use heparan sulfate proteoglycans for it. The successful inhibition of this early event of the infection by synthetic molecules has always been an attractive target for medicinal chemists. Numerous reports have yielded insights into the function of compounds based on the dispirotripiperazine scaffold. Analysis suggests that this is a structural requirement for inhibiting the interactions between viruses and cell-surface heparan sulfate proteoglycans, thus preventing virus entry and replication. This review summarizes our current knowledge about the early history of development, synthesis, structure-activity relationships and antiviral evaluation of dispirotripiperazine-based compounds and where they are going in the future.

Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89's structure may mediate its function.

Targeting the terminase: An important step forward in the treatment and prophylaxis of human cytomegalovirus infections.

A key step in the replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. Enzymes required for this process are so-called terminases, first described for double-stranded DNA bacteriophages. The HCMV terminase consists of the two subunits, the ATPase pUL56 and the nuclease pUL89, and a potential third component pUL51. The terminase subunits are essential for virus replication and are highly conserved throughout the Herpesviridae family. Together with the portal protein pUL104 they form a powerful biological nanomotor. It has been shown for tailed dsDNA bacteriophages that DNA translocation into preformed capsid needs an extraordinary amount of energy. The HCMV terminase subunit pUL56 provides the required ATP hydrolyzing activity. The necessary nuclease activity to cleave the concatemers into unit-length genomes is mediated by the terminase subunit pUL89. Whether this cleavage is mediated by site-specific duplex nicking has not been demonstrated, however, it is required for packaging. Binding to the portal is a prerequisite for DNA translocation. To date, it is a common view that during translocation the terminase moves along some domains of the DNA by a binding and release mechanism. These critical structures have proven to be outstanding targets for drugs to treat HCMV infections because corresponding structures do not exist in mammalian cells. Herein we examine the HCMV terminase as a target for drugs and review several inhibitors discovered by both lead-directed medicinal chemistry and by target-specific design. In addition to producing clinically active compounds the research also has furthered the understanding of the role and function of the terminase itself.

Tetrahalogenated benzimidazole D-ribonucleosides are active against rat cytomegalovirus.

BACKGROUND: Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the viral terminase, the enzyme complex responsible for viral DNA cleavage into single unit-length genomes and subsequent DNA packaging into procapsids. Here, we evaluated the viral inhibition by benzimidazole D-ribonucleosides against rat cytomegalovirus (RCMV). METHODS: Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by measurement of plaque formation. Yield assays and electron microscopy of thin sections was performed using RCMV-infected cells in the presence or absence of the compounds. The effects of Cl4RB and BTCRB on cleavage of concatemers was analyzed by pulsed-field gel electrophoresis. To characterize the behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model was employed where cells are embedded in an extracellular matrix using rat-tail collagen I. RESULTS: Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed that only few virions were formed in RCMV-E infected cells in the presence of the compounds. Pulsed-field gel electrophoresis showed that DNA concatemers failed to be processed in the presence of the compounds. Yield Assays showed a comparable viral growth in the 3D vs. 2D cell culture as well as inhibition in the presence of Cl4RB or BTCRB for RCMV-E/GFP. CONCLUSIONS: These results demonstrate that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV-E by preventing cleavage of concatemeric DNA and nuclear egress of mature capsids.

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

UNLABELLED: DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with ß-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. IMPORTANCE: We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL89 and another postulated packaging protein, pUL93, in infected, as well as transfected, cells.

Kaposi's sarcoma associated herpesvirus tegument protein ORF75 is essential for viral lytic replication and plays a critical role in the antagonization of ND10-instituted intrinsic immunity.

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

DSTP-27 prevents entry of human cytomegalovirus.

Human cytomegalovirus (HCMV) can cause life-threatening diseases in neonates and immunocompromised patients. Due to multiple problems caused by the current available drugs, development of new antiviral compounds is urgently needed. In this study, we characterize the anti-HCMV spectrum and mechanism of action of the N-N'-(bis-5 nitropyrimidyl)dispirotripiperazine derivate 27 (DSTP-27). DSTP-27 exhibited strong antiviral activity against two laboratory HCMV strains with different cell tropism as well as ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates in plaque reduction assays and viral growth kinetics experiments. Interestingly, neither infectious nor noninfectious viral particles were observed by electron microscopy. Pretreatment of cell-free virus with DSTP-27 prevented virus infection. The results from time of addition assays, in which DTSP-27 was added to cells (i) before infection, (ii) during virus adsorption, or (iii) after adsorption, demonstrated an inhibitory effect on early steps of the HCMV replication cycle. This observation was confirmed by immunofluorescence as well as Western blot analysis, whereby reduced levels of the immediate early protein IE1, the processivity factor pUL44, and the tegument protein pp28 were detected. Results from attachment and penetration analyses of prechilled human embryonic lung fibroblasts revealed that virus attachment is not blocked. In addition, DSTP-27 inactivated HCMV by stable binding. Taken together, these results demonstrate that DSTP-27 (i) blocks viral penetration by interacting with the host cell and (ii) inactivates HCMV by interacting with the virus.

Pattern of the epitope-specific IgG/IgM response against human cytomegalovirus in patients with multiple myeloma.

Human cytomegalovirus (HCMV) is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients, e.g., organ transplant recipients and patients with AIDS. One characteristic of herpesviruses is their ability to establish lifelong latency in their hosts; thus, reactivation during immunosuppression leads to recurrent episodes of disease. In several recent reports, it has been shown that HCMV infection may occur in patients with malignancy. This study focused on HCMV infection in patients with multiple myeloma (MM). In order to determine the IgM and IgG humoral immune response, sera from MM patients and healthy donors were analyzed with an accredited immunoblot test, and the IgM response was analyzed with an accredited enzyme-linked immunosorbent assay. A response against HCMV was detected in 80% of the MM patients. While the IgG pattern varied in each patient, the most prominent IgM response was against the tegument protein pp150 and two nonstructural proteins, the processivity factor (pUL44) and the single-stranded DNA binding protein (pUL57). An IgG avidity test revealed that 4 out of 20 MM patients had a fresh infection and 2 MM patients had a recent infection. The combination of IgG avidity and the IgM pattern will be a useful tool for reliable clinical diagnostics concerning HCMV and for application of early therapy for those MM patients suffering from a high viral load.

CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells.

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p<0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

A leucine zipper motif of a tegument protein triggers final envelopment of human cytomegalovirus.

The product of the human cytomegalovirus (HCMV) UL71 gene is conserved throughout the herpesvirus family. During HCMV infection, protein pUL71 is required for efficient virion egress and is involved in the final steps of secondary envelopment leading to infectious viral particles. We found strong indications for oligomerization of pUL71 under native conditions when recombinant pUL71 was negatively stained and analyzed by electron microscopy. Oligomerization of pUL71 during infection was further verified by native and reducing polyacrylamide gel electrophoresis (PAGE). By in silico analyses of the pUL71 sequence, we noticed a basic leucine zipper (bZIP)-like domain, which might serve as an oligomerization domain. We demonstrated the requirement of the bZIP-like domain for pUL71 oligomerization by coimmunoprecipitation and bimolecular fluorescence complementation using a panel of pUL71 mutants. These studies revealed that the mutation of two leucine residues is sufficient to abrogate oligomerization but that intracellular localization of pUL71 was unaffected. To investigate the relevance of the bZIP domain in the viral context, recombinant viruses carrying mutations identical to those in the panel of pUL71 mutants were generated. bZIP-defective viral mutants showed impaired viral growth, a small-plaque phenotype, and an ultrastructural phenotype similar to that of the previously described UL71 stop mutant virus. The majority of virus particles within the viral assembly compartment exhibited various stages of incomplete envelopment, which is consistent with the growth defect for the bZIP mutants. From these data we conclude that the bZIP-like domain is required for oligomerization of pUL71, which seems to be essential for correct envelopment of HCMV.

A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.

Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner.

Short hairpin RNAs specific to human cytomegalovirus terminase subunit pUL89 prevent viral maturation.

BACKGROUND: In order to define the role of the human cytomegalovirus (HCMV) small terminase subunit pUL89, analysis by RNA interference was applied. METHODS: Cell lines expressing pUL89-specific short hairpin RNAs (shRNAs) were constructed by transduction of shRNAs via infection with retroviral vectors. These cell lines were infected with HCMV AD169 and were analysed for pUL89 expression, viral yield, plaque reduction, amount of viral DNA and particle formation. RESULTS: After infection of the cell lines with HCMV, the expression of pUL89 was reduced by up to 86% for shRNA_A and 84% for shRNA_B at the later time points of infection. Cell lines expressing shRNA_C and the control had no effect on the pUL89 expression level. In addition, the inhibitory effect corresponded to a decrease in viral growth kinetics, viral DNA and plaque formation. Analysis by electron microscopy demonstrated that infection of cells expressing pUL89-specific shRNA_A and shRNA_B resulted in a complete inhibition of viral particle formation. CONCLUSIONS: HCMV is a serious life-threatening opportunistic pathogen in immunocompromised patients. Because of multiple problems caused by the current available drugs, development of new strategies are needed. Our data clearly demonstrate that pUL89-specific shRNAs mediated the inhibition of formation of replicative infectious particles and therefore represent a new promising mechanism for antiviral therapy against HCMV infection.

Susceptibilities of human cytomegalovirus clinical isolates and other herpesviruses to new acetylated, tetrahalogenated benzimidazole D-ribonucleosides.

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (Cl(4)RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl(4)RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl(4)RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV- and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCV-sensitive clinical isolates and slightly active against other herpesviruses.

Assembly of monomeric human cytomegalovirus pUL104 into portal structures.

In order for human cytomegalovirus (HCMV) to replicate, concatemeric DNA has to be cleaved into unit-length genomes and packaged into preformed capsids. For packaging to take place and DNA to be translocated, a channel is required in the capsid. Viral capsid channels are generally formed by portal proteins. Here, we show by cross-linking, native gel electrophoresis of infected cells and gel permeation chromatography that the HCMV portal candidate protein pUL104 can form dimers and higher order multimers. Electron microscopy of purified monomeric pUL104 after 5 min incubation revealed that the protein had assembled into a multimeric form and that this form closely resembles complete portal assembly. This is the first study to show that pUL104 monomers have the ability to form portal complexes without additional viral proteins.

Brefeldin A inhibits expression of DNA packaging proteins and nucleocapsid formation of human cytomegalovirus.

In this study we used the fungal antibiotic brefeldin A (BFA) to analyze its effect on viral replication. Analysis by electron microscopy demonstrated that no viral particles were observed in cells treated before the onset of viral replication. In the presence of BFA expression of IE2, MCP, pUL104, pUL56 and pUL89 were reduced, while no or slight effect was observed on expression of pp65, pUL44 and pUL57. Strikingly, real time PCR revealed that de novo viral DNA synthesis is reduced but not completely abolished in the presence of BFA. These results indicated that BFA represents a multi-functional compound leading to inhibition of several steps of viral maturation such as expression of viral DNA packaging proteins and capsid formation.