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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.

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., Author summary HCMV is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients for which the development of new non-nucleosidic antiviral agents are highly important. This manuscript focuses on DNA packaging, which is a target for development of new antivirals. The terminase subunit pUL89 is involved in this process. The paper presents the identification of DNA binding and nuclease motifs with invariant amino acids and highlights its first 3-D surface structure at approx. 3 nm resolution. At this resolution, the calculated 3-D surface structure matches well with the predicted structure. In conjunction with earlier studies it was possible to define structure-function relationships for the HCMV terminase subunit pUL89.

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., Graphical abstract Image 1, Highlights • Several viruses use negatively charged heparan sulfate proteoglycans as entry factors into the host cell. • The binding of these cell-surface features by cationic molecules is a promising antiviral strategy. • Dispirotripiperazines are positively charged spiro compounds first developed as anticancer drugs. • In the following studies, it was found that the compounds also exhibit good antiviral activity. • The review summarizes the existing knowledge about the synthesis and biological activity of dispirotripiperazines.

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.

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 β-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.

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.

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.

To clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles.

The process of viral DNA packaging is multifunctional and determined by specific interactions of protein-DNA and protein-protein. An important role during this process is played by portal proteins. Portals are large macromolecules and are found throughout herpesviruses as well as those double-stranded DNA (dsDNA) bacteriophages examined to date (6). Portal proteins provide on one hand the channel for entry of the DNA during packaging and on the other the exit for releasing DNA during infection. They are incorporated at one unique vertex, a fivefold vertex, of icosahedral procapsids. All known portal proteins differ in size from 35.9 kDa (φ29) to 82.7 kDa (P22) for the monomer, but all are arranged in rings with a 12-fold symmetry (3, 17, 22, 24, 32, 33). Recently, Smith et al. (40) demonstrated that in the case of bacteriophage φ29 the internal force is approximately 50 pN at the end of packaging. Therefore, insertion of dsDNA into the procapsids requires the terminase ATPase activity (39). Portal proteins function, in addition, like a DNA pump as the docking site for the terminase-DNA complex and interact with proteins that seal the portal for preventing DNA loss and reopen it for ejection of the DNA into the nucleus of the host (21). These different functions lead to the suggestion that the portal protein must be comprised of multiple domains and has the ability to change its conformation. Beside the portal the enzymes involved in the packaging process are responsible for site-specific duplex nicking and insertion of the DNA into the procapsids (5, 13), the so-called terminases. The human cytomegalovirus (HCMV) terminase is composed of two subunits, the large one encoding pUL56 and the small pUL89 (7, 8, 9); each protein has a different function. Whereas the large subunit mediates sequence specific DNA binding and ATP hydrolysis, pUL89 is only required for duplex nicking (23, 36, 37). The hydrolysis of ATP has multiple functions during the packaging process. It is also involved in the formation of the packaging complex. The anti-HCMV benzimidazole-d-nucleosides, BDCRB and TCRB, developed in Townsend's and Drach's laboratories (43) target the HCMV terminase. With Biron and coworkers, they showed for the first time that the HCMV maturation was inhibited by these compounds (27, 44). By marker transfer experiments, mutations in UL89 (44) as well as UL56 (26) were found. Unlike ATP hydrolyzing terminase subunits of most other icosahedral virions, e.g., dsDNA bacteriophages and herpes simplex virus type 1 (HSV-1), the large subunit pUL56 is stably associated with the capsid and represents a structural component (4, 14, 38, 47). Only one terminase protein, P9 of bacteriophage PRD1, is also a structural component of the virion (31, 42). Recently, we showed by three-dimensional reconstruction that pUL56 forms a dimer with a C-2 symmetry (35). This structure is the prerequisite for the formation of a protein-DNA complex required for packaging into the procapsid. The first herpesvirus portal protein encoded by open reading frame (ORF) UL6 of HSV-1 (11, 29, 34) was identified by Newcomb et al. (32). Recently, we could demonstrate that the HCMV ORF UL104 encodes a protein with an Mr of approximately 73,000 with a tendency to dimerize. Furthermore, pUL104 has the ability to bind sequence-independent to double-stranded DNA. Under native conditions pUL104 assembles into high-molecular-weight complexes (15). In this study, experiments are described concerning the colocalization of pUL104 with packaging proteins and a direct physical interaction regarding the HCMV large terminase subunit pUL56 and its inhibition by benzimidazole-d-ribonucleosides.

The matrix protein M of Borna disease virus (BDV) is a constituent of the viral envelope covering the inner leaflet of the lipid bilayer. BDV-M was expressed as recombinant protein in Escherichia coli, purified to homogeneity and structurally analyzed. Recombinant M (i) forms non-covalently bound multimers with a Stoke's radius of 35 Angstroms estimated by size exclusion chromatography, (ii) consists of tetramers detected by analytical ultracentrifugation, and (iii) appears by electron microscopy studies as tetramers with the tendency to assemble into high molecular mass lattice-like complexes. The structural features suggest that BDV-M possesses a dominant driving force for virus particle formation.

Herpesviral DNA packaging is a complex process involving binding and cleavage of DNA containing the specific DNA-packaging motifs, pac1 and pac2, and packaging of the resulting unit-length genomes into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of human cytomegalovirus the terminase consists of the proteins pUL56 and pUL89. While pUL56 (i) mediates the specific binding to pac sequences on the concatamers, (ii) provides energy for the translocation of the DNA to the procapsids and (iii) associates itself with the capsid for enabling the entry of the DNA into the procapsid, pUL89 is mainly required to effect DNA cleavage. Based on the limited efficacy of the current drugs ganciclovir, cidofovir and foscarnet, new antiviral therapeutics appear to be in demand. Inhibitors targeting pUL56 and/or pUL89 may offer an attractive alternative since mammalian cell DNA replication does not involve cleavage of concatameric DNA. Drugs targeted to terminase-like proteins should therefore be safe and highly selective.

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., Author Summary Kaposi's Sarcoma-Associated Herpesvirus (KSHV) establishes a lifelong persistent infection in humans and is associated with tumors and lymphoproliferative disease, particularly upon immunosuppression. The virus has to overcome cellular intrinsic immunity in order to initiate viral protein expression and genome replication in primary infection. We demonstrated that KSHV is restricted by a cellular intrinsic immunity complex called nuclear domain 10 (ND10) and identified a critical role of the KSHV ORF75 protein, which is part of the viral particle, in this process. We found that ORF75 is essential for viral replication and that ORF75 leads to disappearance of the ND10 protein ATRX. Furthermore, it induces the relocalization of several other ND10 components. Noteworthy, all herpesviruses studied so far have evolved mechanisms for ND10 counteraction, indicating the importance of this step for herpesviral replication. The individual mechanisms, however, including the extent of ND10-antagonization, are of considerable variation between different herpesviruses. We speculate that, in contrast to efficient lytic replication of alphaherpesviruses, less effective ND10 counteraction may represent a doorway for gammaherpesviruses to latent infection.

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.

Permanent cell lines showing homogeneous constitutive expression of glycoprotein B (gpUL55; gB) of human cytomegalovirus (HCMV) were selected, in the presence of geneticin, from human astrocytoma cells (U373) after transfection with recombinant pRC/CMV-gB carrying the complete coding sequence for HCMV gB and for aminoglycoside phosphotransferase. The biosynthesis and processing including specific proteolytic cleavage, formation of disulphide-linked oligomers as well as transport of recombinant gB in three of four established transformed cell lines essentially resembled that found in infected parental U373 except for eventual degradation after 2 h of gB synthesis. Analysis of the fourth transformant expressing uncleaved gB suggested that proteolytic cleavage is not required for normal intracellular transport. The stable transformants retained permissiveness for productive superinfection with HCMV. The application of cell lines transformed with mutagenized HCMV gB for the rescue of genetically engineered HCMV mutants is discussed.

Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri- O -acetyl-β- d -ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri- O -acetyl-β- 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.

Human cytomegalovirus (HCMV) is a member of the herpesvirus family and represents a major human pathogen causing life-threatening diseases. HCMV persists lifelong in its host because the immune system does not achieve clearance of the virus. Since the currently available anti-HCMV drugs cause multiple problems new antiviral therapeutics are urgently in demand. The ubiquitin-proteasome system (UPS) not only mediate the degradation of mis-folded proteins but is also involved in processes essential for viral replication. Thus the UPS could represent a new target for anti-HCMV therapy supporting a different mode of action. In this chapter we will review the impact of proteasome inhibition on HCMV replication and discuss whether the use of proteasome inhibitors is a possible strategy to prevent viral replication.

Contents Part 1. Concepts of therapy for RNA viruses Therapeutic vaccination in chronic hepadnavirus infection by Michael Rogendorf and Mengji Lu Characterization of targets for antiviral therapy of Flaviridae infections by Peter Borowski Inhibition of Hepatitis C virus by nucleic acid-based antiviral approaches by Michael Frese and Ralf Bartenschlager Inhibitors for respiratory viruses by Ernst Kuechler and Joachim Seipelt Anti-viral approaches against influenza viruses by Stephan Pleschka, Stephan Ludwig, Thorsten Wolff and Oliver Planz A new approach to an Influenza virus live vaccine: Modification of the cleavage site of the haemagglutine by reverse genetics by Jurgen Stech, Holger Garn and Hans-Dieter Klenk New concepts in anti-HIV therapies by Justin Stebbing, Mark Bower and Brian Gazzard Evaluation of current strategies to inhibit HIV entry, integration and maturation by Jaqueline D. Reeves, Stephen D. Barr and Stephan Poehlmann Managing antiretroviral resistance by Barbara Schmidt, Monika Tschoschner, Hauke Walter and Klaus Korn Part 2. Concepts of therapy for DNA viruses Selective inhibitors of the replication of poxviruses by Johan Neyts and Erik de Clercq Maribavir: A promising new antiherpes therapeutic Agent by Karen K. Biron Benzimidazle-D-ribonucleosides as antiviral agents that target HCMV terminase by John C. Drach, Leroy B. Townsend and Elke Bogner Recent developments in anti-herpesviral therapy based on protein kinase inhibitors by Thomas Herget and Manfred Marschall Immune therapy against papillomavirus-related tumors in humans by Lutz Gissmann Part 3. Concepts of therapy for emerging viruses The SARS Corona virus receptor ACE-2 A potential target for antiviral therapy by Jens H. Kuhn, Sheli R. Radoshitzky, Wenhui Li, Swee Kee Wong, Hyeryun Choe and Michael Farzan Therapy of Ebola and Marburg virus infections by MikeBray Part 4. General concepts of therapy Proteasome inhibitors as complementary or alternative antiviral therapeutics by Susanna Proesch and Marion Kaspari Human monoclonal antibodies for prophylaxis and therapy of viral infections by Jan ter Meulen and Jaap Goudsmit Vector-based antiviral therapy by Armin Ensser Index

Herpesviruses have large (130-235 kb) double-stranded linear DNA genomes that circularise shortly after infection (Garber et al., 1993; McVoy and Adler, 1994; Roizman and Sears, 1996). During infection, viral DNA is replicated in nuclei of infected cells and accumulates as head-to-tail concatemers (Ben-Porat, 1983; Jacob et al., 1979; Martinez et al., 1996; McVoy and Adler, 1994; Roizman and Sears, 1996; Severini et al., 1994; Zhang et al., 1994). Unit-length genomes are cleaved from intranuclear concatemeric viral DNA and packaged into preassembled capsids. This process is the first essential step in the production of infectious particles, i.e. virions. Considering that current anti-herpesviral drugs like acyclovir, ganciclovir, cidofovir and foscarnet have limited effects and often exhibit dose-dependent toxicity, new antiviral therapeutics ought to be considered. This, in turn, would necessitate the characterisation of new targets supporting different modes of action. To this end, an active interference with the cleavage and packaging mechanisms of the viral DNA may offer viable alternatives. This chapter is exclusively concerned with the packaging of the viral genome into the procapsid. For a review of the assembly mechanisms of capsids and procapsids from their proteinaceous building blocks, the reader is referred to chapter 3.1.

1. Cell entry and egress. 1.1. Epstein-Barr Virus Glycoproteins and their Roles in Virus Entry and Egress L. Hutt-Fletcher. 1.2. Structure and Function of Viral Glycoproteins in Membrane Fusion W. Weissenhorn. 2. Viral replication. 2.1. Viral RNA-directed Polymerases: Structure and Function Relationships J. Jager, J.D. Pata. 2.2. Marburg Virus Replication and Nucleocapsid Formation: Different Jobs, Same Players E. Muhlberger, S. Becker. 3. Viral determinants for capsid formation and packaging. 3.1. Packaging DNA into Herpesvirus Capsids J.C. Brown, et al. 3.2. From Concatemeric DNA into Unit-length Genomes - a Miracle or Clever Genes? A. Holzenburg, E. Bogner. 4. Determinants for viral maturation. 4.1. Role of Envelope Proteins in Measles Virus Assembly A. Maisner. 4.2. The Predominant Varicella-zoster Virus gE and gI Glycoprotein Complex C. Grose. 4.3. Expression Strategy and Functions of the Filoviral Glycoproteins V.E. Volchkov, H. Feldman. 5. Pathogenesis. 5.1. Prions: When Proteins Take a Wrong Turn A. Bellon, M. Vey. 5.2. Human Immunodeficiency Virus: From Virus Structure to Pathogenesis H. Gelderblom, K. Boller. 5.3. The role of Hemagglutinin and Neuraminidase in Influenza Virus Pathogenesis R. Wagner, et al. 6. Viral oncogenesis. 6.1. Rhadinovirus Pathogenesis A. Ensser, et al. 6.2. Interaction of Papillomaviral Oncoproteins with Cellular Factors S. Smola-Hess, H.J. Pfister. 7. Defense mechanisms. 7.1. Cytomegaloviral Glycoprotein Interacting with MHC Class I Moleculesand the MHC-encoded Peptide Transporter A. Zimmermann, et al. 7.2. Regulation of Cellular Genes by Cytomegalovirus M.F. Stinski, Yoon-Jae Song. Index.

Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be involved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence analysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucleus of infected cells. Solitary expression of wild-type as well as epitope-tagged pUL56 also resulted in nuclear distribution after transfection, suggesting the presence of an endogenous nuclear localization signal (NLS). Deletion of a carboxy-terminal stretch of basic amino acids (aa 816–827) prevented nuclear translocation, indicating that the sequence RRVRATRKRPRR of HCMV pUL56 mediates nuclear targetting. The signal character of the NLS sequence was demonstrated by successful transfer of the NLS to a reporter protein chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions within the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation. In order to identify the transport machinery involved, the potential of pUL56 to bind importin α (hSRP1α) was examined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1α was provided byin vitrobinding assays. In view of these findings, it is suggested that nuclear translocation of HCMV pUL56 is mediated by the importin-dependent pathway.

Surface biotinylation of human cytomegalovirus (HCMV)-infected fibroblasts under pulse-chase conditions was used to define the cellular route of the dominant viral envelope glycoprotein gB into the cytoplasmic compartment of viral maturational envelopment. The results showed that a major fraction of gB was re-internalized from the infected cell surface prior to incorporation into the viral envelope. Viral particles carrying biotinylated gB were subsequently released into the culture medium. Viral release appeared to be inhibited in the presence of gB-specific antibody or when infected cultures were incubated at room temperature, but was not reduced by inhibitors of cellular glycoprotein transport. To our knowledge this is the first report describing that HCMV gB is retrieved from the infected cell surface prior to viral envelopment.

Experiments were undertaken to identify the product of open reading frame (orf) UL 56 of human cytomegalovirus (HCMV), the putative homolog of infected cell protein (ICP) 18.5 of herpes simplex virus type 1 (HSV- 1) that is thought to be involved in viral nucleocapsid maturation. Northern blotting using a unique fragment of orf UL56 revealed a specific transcript of about 3.0 kb in HCMV-infected fibroblasts early and late postinfection (p.i.). Two overlapping fragments of UL56 were subcloned for procaryotic expression and the recombinant proteins were used for affinity purification of an antibody fraction from human convalescent serum (pab UL56). The affinity purified antibody recognized a polypeptide with an apparent molecular weight of about 130 kDa (p 130) in immunoblots of virions and of infected cell extracts late p.i. Upon treatment of isolated virions with nonionic detergent the p 130 polypeptide separated with the nucleocapsid/tegument fraction. The amino acid sequence of p 130 deduced from the nucleotide sequence exhibited a high homology to ICP 18.5 of HSV-1.

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.

The highly conserved DNA-binding protein pUL56 of human cytomegalovirus (HCMV) was found to be predominantly localized throughout the nucleus as well as in viral replication centers of infected cells. The latter localization was abolished by phosphono acetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL56 co-localized in replication centers alongside pUL112–113 and pUL44 at late times of infection. By co-immunoprecipitations, a direct interaction with pUL44, a protein of the replication fork, was detected. These results showed for the first time that HCMV pUL56 is localized in viral replication centers, implicating that DNA replication is coupled with packaging.