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Autophagy is an important component of the innate immune response that restricts infection by different types of pathogens. Viruses have developed multiple strategies to avoid autophagy to complete their replication cycle and promote spreading to new hosts. Here, we report that the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autophagy by inhibiting the activity of the autophagy receptor SQSTM1/p62. We found that all the homologs bind to and deubiquitinate SQSTM1/p62 but with variable efficiency, which correlates with their capacity to prevent the colocalization of light chain 3 (LC3) with SQSTM1/p62 aggregates and promote the accumulation of a model autophagy substrate. The findings highlight important differences in the strategies by which herpesviruses interfere with selective autophagy., (© 2021 The Author(s).)

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of β-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.

Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 10 1.2 copies/µl and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.

Herpesvirus infection is an orderly, regulated process. Among these viruses, the encapsidation of viral DNA is a noteworthy link; the entire process requires a powered motor that binds to viral DNA and carries it into the preformed capsid. Studies have shown that this power motor is a complex composed of a large subunit, a small subunit, and a third subunit, which are collectively known as terminase. The terminase large subunit is highly conserved in herpesvirus. It mainly includes two domains: the C-terminal nuclease domain, which cuts the viral concatemeric DNA into a monomeric genome, and the N-terminal ATPase domain, which hydrolyzes ATP to provide energy for the genome cutting and transfer activities. Because this process is not present in eukaryotic cells, it provides a reliable theoretical basis for the development of safe and effective anti-herpesvirus drugs. This article reviews the genetic characteristics, protein structure, and function of the herpesvirus terminase large subunit, as well as the antiviral drugs that target the terminase large subunit. We hope to provide a theoretical basis for the prevention and treatment of herpesvirus.

Herpesviruses thymidine kinase (TK) and protein kinase (PK) allow the activation of nucleoside analogues used in anti-herpesvirus treatments. Mutations emerging in these two genes often lead to emergence of drug-resistant strains responsible for life-threatening diseases in immunocompromised populations. In this review, we analyze the binding of different nucleoside analogues to the TK active site of the three α-herpesviruses [Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) and Varicella-Zoster Virus (VZV)] and present the impact of known mutations on the structure of the viral TKs. Furthermore, models of β-herpesviruses [Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6)] PKs allow to link amino acid changes with resistance to ganciclovir and/or maribavir, an investigational chemotherapeutic used in patients with multidrug-resistant HCMV. Finally, we set the basis for the understanding of drug-resistance in γ-herpesviruses [Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV)] TK and PK through the use of animal surrogate models., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pU L 15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pU L 15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pU L 15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pU L 15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pU L 15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. The proteins are expressed during the productive virus cycle and are incorporated into infectious virus particles, being delivered to the target cells upon primary infection. Members of this viral enzyme family were shown to regulate different aspects of the virus life cycle and the innate anti-viral response. However, only few substrates have been identified and the mechanisms of these effects remain largely unknown. In order to gain insights on the substrates and signaling pathways targeted by the viral enzymes, we have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with the Epstein-Barr virus encoded homologue BPLF1. Several members of the 14-3-3-family of scaffold proteins were found amongst the top hits of the BPLF1 interactome, suggesting that, through this interaction, BPLF1 may regulate a variety of cellular signaling pathways. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a tri-molecular complex including, in addition to 14-3-3, the ubiquitin ligase TRIM25 that participates in the innate immune response via ubiquitination of cytosolic pattern recognition receptor, RIG-I. The involvement of BPLF1 in the regulation of this signaling pathway was confirmed by inhibition of the type-I IFN responses in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that the active viral enzyme promotes the dimerization and autoubiquitination of TRIM25. Upon triggering of the IFN response, RIG-I is recruited to the complex but ubiquitination is severely impaired, which functionally inactivates the RIG-I signalosome. The capacity to bind to and functionally inactivate the RIG-I signalosome is shared by the homologues encoded by other human herpesviruses.

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes., (Published by Elsevier Inc.)

During assembly of herpesvirus capsids, a protein scaffold self-assembles to ring-like structures forming the scaffold of the spherical procapsids. Proteolytic activity of the herpesvirus maturational protease causes structural changes that result in angularization of the capsids. In those mature icosahedral capsids, the packaging of viral DNA into the capsids can take place. The strictly regulated protease is called assemblin. It is inactive in its monomeric state and activated by dimerization. The structures of the dimeric forms of several assemblins from all herpesvirus subfamilies have been elucidated in the last two decades. They revealed a unique serine-protease fold with a catalytic triad consisting of a serine and two histidines. Inhibitors that disturb dimerization by binding to the dimerization area were found recently. Additionally, the structure of the monomeric form of assemblin from pseudorabies virus and some monomer-like structures of Kaposi's sarcoma-associated herpesvirus assemblin were solved. These findings are the proof-of-principle for the development of new anti-herpesvirus drugs. Therefore, the most important information on this fascinating and unique class of proteases is summarized here.

The antiherpetic drugs acyclovir (ACV, valaciclovir) and penciclovir (famciclovir) are phosphorylated by viral thymidine kinase and terminate DNA synthesis. ASP2151 (amenamevir) and foscavir (PFA) directly inhibit viral helicase-primase and DNA polymerase, respectively, and inhibit replication of herpes simplex virus (HSV) and varicella-zoster virus. ACV, ASP2151, and PFA all inhibit HSV with a different mechanism of action and as a consequence, the kinetics of viral DNA accumulation and progeny virus production differ. This study focused on how viral DNA synthesis and its related events in the replication cycle would influence anti-HSV action of ACV, ASP2151, and PFA. ASP2151 suppressed HSV replication more efficiently than ACV at 10 × 50% effective concentration of plaque formation (EC 50 ), when treatments were started 0-24 h after infection. ASP2151 and PFA were more potent than ACV in suppressing viral DNA synthesis and infectious virus production when they were added up to 3 h following infection. The virus replicated in the presence of ACV was compared for the ratios of HSV DNA copy number to infectivity with that without ACV and infectivity of ACV-treated virus was less efficient than that without ACV-treatment. The EC 50 of infected cells in the time course after infection was preserved in PFA, limited in ASP2151, and much increased for ACV, indicating that viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but reduced the susceptibility of ACV. ASP2151 showed a preferable profile as an anti-herpetic agent with a better pharmacokinetic profile than ACV., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Many large viruses, including tailed dsDNA bacteriophages and herpesviruses, assemble their capsids via formation of precursors, called procapsids or proheads. The prohead has an internal core, made of scaffolding proteins, and an outer shell, formed by the major capsid protein. The prohead usually contains a protease, which is activated during capsid maturation to destroy the inner core and liberate space for the genome. Here, we report a 2.0 Å resolution structure of the pentameric procapsid protease of bacteriophage T4, gene product (gp)21. The structure corresponds to the enzyme's pre-active state in which its N-terminal region blocks the catalytic center, demonstrating that the activation mechanism involves self-cleavage of nine N-terminal residues. We describe similarities and differences between T4 gp21 and related herpesvirus proteases. We found that gp21 and the herpesvirus proteases have similarity with proteins forming the tubes of phage tails and bacterial type VI secretion systems, suggesting their common evolutionary origin., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Herpesviruses have been reported in several marsupial species, but molecular classification has been limited to four herpesviruses in macropodids, a gammaherpesvirus in two antechinus species (Antechinus flavipes and Antechinus agilis), a gammaherpesvirus in a potoroid, the eastern bettong (Bettongia gaimardi) and two gammaherpesviruses in koalas (Phascolarctos cinereus). In this study we examined a range of Australian marsupials for the presence of herpesviruses using molecular and serological techniques, and also assessed risk factors associated with herpesvirus infection. Our study population included 99 koalas (Phascolarctos cinereus), 96 eastern grey kangaroos (Macropus giganteus), 50 Tasmanian devils (Sarcophilus harrisii) and 33 common wombats (Vombatus ursinius). In total, six novel herpesviruses (one alphaherpesvirus and five gammaherpesviruses) were identified in various host species. The overall prevalence of detection of herpesvirus DNA in our study population was 27.2% (95% confidence interval (CI) of 22.6-32.2%), but this varied between species and reached as high as 45.4% (95% CI 28.1-63.7%) in common wombats. Serum antibodies to two closely related macropodid herpesviruses (macropodid herpesvirus 1 and 2) were detected in 44.3% (95% CI 33.1-55.9%) of animals tested. This also varied between species and was as high as 92% (95% CI 74.0-99.0%) in eastern grey kangaroos. A number of epidemiological variables were identified as positive predictors for the presence of herpesvirus DNA in the marsupial samples evaluated. The most striking association was observed in koalas, where the presence of Chlamydia pecorum DNA was strongly associated with the presence of herpesvirus DNA (Odds Ratio = 60, 95% CI 12.1-297.8). Our results demonstrate the common presence of herpesviruses in Australian marsupials and provide directions for future research.

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Introduction: The standard therapy used to treat herpesvirus infections is based on the application of DNA polymerase inhibitors such as ganciclovir or aciclovir. Unfortunately, all of these compounds exhibit relatively high toxicity and the mutation of herpesviruses results in the appearance of new drug-resistant strains. Consequently, there is a great need for the development of new, effective and safe anti-herpesvirus agents that employ different patterns of therapeutic action at various stages of the virus life cycle., Areas Covered: Patents and patent applications concerning the development of anti-herpesvirus agents displaying different mechanisms of action that have been published since 2003 are reviewed. In addition, major discoveries in this field that have been published in academic papers have also been included., Expert Opinion: Among all the anti-herpesvirus agents described in this article, the inhibitors of viral serine protease seem to present one of the most effective/promising therapeutics. Unfortunately, the practical application of these antiviral agents has not yet been proven in any clinical trials. Nevertheless, the dynamic and extensive work on this subject gives hope that a new class of anti-herpesvirus agents aimed at the enzymatic activity of herpesvirus serine protease may be developed.

The HindIII-HincII fragment of the 5.5 kbp H11 HindIII clone of ovine herpesvirus 1 (OvHV-1) was cloned and its primary structure was determined by preparation of nested deletion subclones and their sequencing. Sequence analysis of the overlapping clones revealed that 3239 bp OvHV-1 fragment contains complete thymidine kinase (TK) gene, a partial open reading frame of ORF20 and that encoding glycoprotein H (gH). The conserved OvHV-1 TK displayed the highest similarity to homologous TK proteins encoded by members of the Macavirus genus of the Gammaherpesvirinae subfamily. These data including our previous analysis of the partial sequence of VP23 homologue might serve as further evidence that OvHV-1 should be categorized within the genus Macavirus of the Herpesviridae family.

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Herpesviruses are ubiquitous human pathogens that establish lifelong persistent infections. Clinical manifestations range from mild self-limiting outbreaks such as childhood rashes and cold sores to the more severe and life-threatening outcomes of disseminated infection, encephalitis, and cancer. Nucleoside analog drugs that target viral DNA replication provide the primary means of treatment. However, extended use of these drugs can result in selection for drug-resistant strains, particularly in immunocompromised patients. In this review we will present recent observations about the participation of cellular protein kinases in herpesvirus biology and discuss the potential for targeting these protein kinases as well as the herpesvirus-encoded protein kinases as an anti-herpesvirus therapeutic strategy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Viral polymerases are important targets in drug discovery and development efforts. Most antiviral compounds that are currently approved for treatment of infection with members of the herpesviridae family were shown to inhibit the viral DNA polymerase. However, biochemical studies that shed light on mechanisms of drug action and resistance are hampered primarily due to technical problems associated with enzyme expression and purification. In contrast, the orthologous bacteriophage RB69 polymerase gp43 has been crystallized in various forms and therefore serves as a model system that provides a better understanding of structure-function relationships of polymerases that belong the type B family. This review aims to discuss strengths, limitations, and opportunities of the phage surrogate with emphasis placed on its utility in the discovery and development of anti-herpetic drugs.

Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.

Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cellular protein synthesis machinery is tightly regulated and capable of rapid reaction to a variety of physiological inputs critical in stress-response, cell cycle control, cancer biology, and virus infection. One important strategy for stimulating protein synthesis involves the ser/thr kinase Akt, which subsequently triggers inactivation of the cap-dependent translational repressor 4E-BP1 by an mTOR-containing protein complex (mTORC1). A recent paper demonstrated that herpes simplex virus utilizes a remarkable tactic to activate mTOR in infected cells. Instead of using the cellular Akt, the virus produces a ser / thr kinase called Us3 that doesn't look like Akt, but masquerades as Akt. By making the Akt-like protein unrecognizable, this disguise allows it to bypass the strict limits normally imposed on the real cellular Akt. Importantly, preventing the virus Akt-imposter from triggering mTORC1 inhibited viral growth, suggesting a new way to block herpes simplex virus. This study also raises the possibility that other Akt-impersonators may lurk hidden in our own genomes, possibly contributing to diseases ranging from diabetes to cancer.

Koi herpesvirus (KHV) causes a fatal disease in koi and common carp, but no reliable and genetically characterized vaccines are available up to now. Therefore, we generated KHV recombinants possessing deletions within the viral ribonucleotide reductase (RNR), thymidine kinase (TK), dUTPase, or TK and dUTPase genes, and their corresponding rescuants. All KHV mutants were replication competent in cultured cells. Whereas plaque sizes and titers of RNR-negative KHV were reduced, replication of the other mutants was not affected. Experimental infection of carp indicated attenuation of TK- or dUTPase-deleted KHV, and PCR analysis of tissue samples permitted differentiation of mutant from wild-type virus.

Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4(+) T cells from cord blood that have undetectable levels of the eight human herpesviruses. Additionally, acyclovir phosphates were detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified in a primer extension assay from cord blood. The data support acyclovir as an inhibitor of HIV-1 replication in herpesvirus-negative cells.

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus., (Copyright 2010. Published by Elsevier Inc.)

The low toxicity of acyclovir (ACV) is mainly due to the fact that human nucleoside kinases have undetectable phosphorylation rates with this acyclic guanine analogue. In contrast, herpes virus thymidine kinase (HSV1-TK) readily activates ACV. We wanted to understand why human deoxycytidine kinase (dCK), which is related to HSV1-TK and phosphorylates deoxyguanosine, does not accept acyclic guanine analogues as substrates. Therefore, we crystallized dCK in complex with ACV at the nucleoside phosphoryl acceptor site and UDP at the phosphoryl donor site. The structure reveals that while ACV does bind at the dCK active site, it does so adopting a nonproductive conformation. Despite binding ACV, the enzyme remains in the open, inactive state. In comparison to ACV binding to HSV1-TK, in dCK, the nucleoside base adopts a different orientation related by about a 60 degrees rotation. Our analysis suggests that dCK would phosphorylate acyclic guanine analogues if they can induce a similar rotation.

A series of C-2 pyrroloquinoline analogs designed to improve aqueous solubility were examined for herpesvirus polymerase and antiviral activity. Several analogs were identified that maintained the antiviral activity of the previous development candidate against HCMV, HSV-1 and VZV, but with significantly improved aqueous solubility., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Tumor-specific promoters are predominantly active and ensure expression of the gene under control exclusively in cancer cells. However, a low activity of the promoters is an essential disadvantage for their therapy usage. To achieve a higher expression level of the therapeutic gene, herpes simplex virus thymidine kinase (HSV-tk), the Tat-TAR-system being utilized by HIV-1 for increasing own gene expression was developed. A potentiating activity of tat gene under control of two different cancer-specific gene (human survivin gene and human telomerase reverse transcriptase) promoters for increasing of the HSV-tk gene expression being regulated by TAR-element was evaluated, and activity of the cancer-specific promoters in the Tat-TAR-system was compared. Co-transfection of the cells with the both constructions led to the tat protein synthesis and its affect the HIV-1 TAR-element. An expression level of HSV-tk gene ensured by the both promoters in the binary system was close to that for strong non-specific cytomegalovirus (CMV) promoter. Enzymatic activity of HSV-tk protein in cells having both elements of Tat-TAR-system was two orders of magnitude higher than that in the cells transfected with HSV-tk gene under control of the cancer-specific promoter. Notably, the effect was independent of p53-status of transfected cells: HSV-tk expression level was almost the same in p53(+) and p53(-) cells. The obtained results show that system may be used for therapy of different cancer types both p53-defective and p53-positive ones inhibiting cancer-specific promoters activity.

Ribonucleotide reductase (RNR), a crucial enzyme for nucleotide anabolism, is encoded by all living organisms and by large DNA viruses such as the herpesviruses. Surprisingly, the beta-herpesvirus subfamily RNR R1 subunit homologues are catalytically inactive and their function remained enigmatic for many years. Recent work sheds light on the function of M45, the murine cytomegalovirus R1 homologue; during viral evolution, M45 apparently lost its original RNR activity but gained the ability, via inhibiting RIP1, a cellular adaptor protein, to block cellular signaling pathways involved in innate immunity and inflammation. The discovery of this novel mechanism of viral immune subversion provides further support to the concept of evolutionary tinkering.

Axonal transport is essential for the successful establishment of neuroinvasive herpesvirus infections in peripheral ganglia (retrograde transport) and the subsequent spread to exposed body surfaces following reactivation from latency (anterograde transport). We examined two components of pseudorabies virus (US3 and UL13), both of which are protein kinases, as potential regulators of axon transport. Following replication of mutant viruses lacking kinase activity, newly assembled capsids displayed an increase in retrograde motion that prevented efficient delivery of capsids to the distal axon. The aberrant increase in retrograde motion was accompanied by loss of a viral membrane marker from the transported capsids, indicating that the viral kinases allow for efficient anterograde transport by stabilizing membrane-capsid interactions during the long transit from the neuron cell body to the distal axon.

The thymidine kinases from feline herpesvirus (FHV TK) and canine herpesvirus (CHV TK) were cloned and characterized. The two proteins are closely sequence-related to each other and also to the herpes simplex virus type 1 thymidine kinase (HSV-1 TK). Although FHV TK and CHV TK have a level of identity of 31 and 35%, respectively, with HSV-1 TK, and a general amino acid similarity of approximately 54% with HSV-1 TK, they do not recognize the same broad range of substrates as HSV-1 TK does. Instead the substrate recognition is restricted to dThd and pyrimidine analogs such as 1-beta-d-arabinofuranosylthymine (araT), 3'-azido-2',3'-dideoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). FHV TK and CHV TK differ in substrate recognition from mammalian cytosolic thymidine kinase 1 (TK1) in that TK1 does not phosphorylate BVDU and they also differ from mammalian mitochondrial thymidine kinase 2 (TK2), which, in addition to thymidine and thymidine analogs also phosphorylates dCyd. Although the nucleoside analog BVDU was a good substrate for FHV and CHV TK, the compound was poorly inhibitory to virus-induced cytopathic effect in FHV- and CHV-infected cells. The reason is likely the poor, if any, thymidylate kinase activity of FHV and CHV TK, which in HSV-1 TK-expressing cells convert BVDU-MP to its 5'-diphosphate derivative.

Initial phylogenetic comparisons based on a region of the DNA polymerase of seven herpes-like viruses found in sturgeons in North America and Europe indicated the presence of three distinct clades. A revised phylogenetic analysis of the same viruses, based on corrected DNA polymerase sequences and newly obtained sequence data from the putative ATP subunit of the terminase gene, indicate only two clades. These two clades correspond to the historical designations given to these herpes-like viruses from white sturgeon Acipenser transmontanus: white sturgeon herpesvirus type 1 (WSHV-1) and type 2 (WSHV-2). The identification of putative terminase gene sequences for all seven herpes-like viruses from sturgeons confirms their affinity with the family Herpesviridae (because this gene is unique to herpesviruses) and more distantly with T4-like bacteriophages. The two clades of sturgeon herpesviruses are therefore appropriately designated as Acipenserid herpesviruses 1 and 2, which correspond to the previous common names of white sturgeon herpesvirus types 1 and 2.

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however, along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task.

The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Marek's disease virus (MDV), a tumorigenic alphaherpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant outgrowths.

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Herpesviral protein kinases of the UL97 subfamily are expressed by all known herpesviruses but the degree of functional conservation is unclear. A selection of representative members was investigated by a comparative structural and functional analysis. The coding sequences of human cytomegalovirus (HCMV) pUL97, rat CMV pR97, Epstein-Barr virus BGLF4, and herpes simplex virus UL13 showed a low degree of amino acid identity. A computational approach employing fold recognition techniques revealed structural similarity to the cellular kinase Cdk2 with a high level of conservation of the functionally important residues in ATP binding sites and the catalytic centers. Analyses of in vitro activities of these herpesviral protein kinases, including measurements of phosphorylation of cellular substrates, trans-complementation experiments with a UL97-deleted HCMV mutant, and sensitivity profiles toward protein kinase inhibitors, demonstrated marked similarities between pUL97 and pR97 and to a lesser extent between pUL97 and BGLF4 or UL13. Thus, the structure-activity analysis of pUL97-like herpesviral protein kinases indicates a partial but not a full conservation of their functional properties among the herpesviruses.

Endotheliotropic elephant herpesvirus (elephantid herpesvirus 1; ElHV-1) is apathogenic for African elephants (Loxodonta africana), but causes fatal haemorrhagic disease in Asian elephants (Elephas maximus). This is thought to occur through transmission from African elephants in places where both species are housed, such as zoological gardens. The virus has caused considerable losses in North American and European zoological gardens and thus severely impedes breeding of the endangered Asian elephant. Previously, the ultrastructural and genetic characterization of ElHV-1 from a male Asian elephant that died from the disease at the Berlin zoological gardens in 1998 have been reported. Here, a partial characterization of the ElHV-1 genome is presented. A 60 kbp locus, spanning 34 open reading frames, was analysed. Most of the detected genes were found to be conserved among the herpesviruses and showed an overall arrangement most similar to that of betaherpesviruses, in particular Human herpesvirus 6 and Human herpesvirus 7. Most importantly, in addition to a protein kinase gene that is homologous to the human cytomegalovirus UL97 gene, a thymidine kinase (TK) gene was found, which is generally missing in betaherpesvirus genomes. Thus, ElHV-1 is the only known betaherpesvirus to encode a TK gene. This peculiarity might contribute to the fulminant pathogenicity of ElHV-1, but also provide a crucial enzymic activity for developing an efficient antiviral therapy with currently available nucleoside analogues.

Viral DNA and RNA polymerases are enzymes, which are responsible for copying the genetic materials of viruses and are therefore central components in the life cycles of viruses. The polymerases are essentially required for the replication of viruses. The reverse transcriptase (RT) of the retroviruses and the hepadnaviruses is the sole viral enzyme required for the synthesis of DNA from viral RNA. Viral polymerases are therefore an extremely favorable target for the development of antiviral therapy. The success of anti-HIV-1 therapy using inhibitors specifically targeting HIV RT suggests that other viral polymerases can be the valid molecular targets for the design of antiviral drugs. Intensive structural and functional studies of viral polymerases have been conducted and have opened new avenues for the development of more effective antiviral therapy. This review summarizes the insights gained from recent structural and functional studies of antiviral agents, which target viral polymerases. The primary focus will be on hepatitis C virus (HCV), herpesviruses, HIV, hepatitis B virus (HBV) and influenza virus.

Proteases fulfill multiple roles in health and disease, and considerable interest has been expressed in the design and development of synthetic inhibitors of disease-related proteases. Virus-encoded proteases have been shown to be involved in the replication of many viruses. The success of anti-HIV-1 therapy using specific and potent protease inhibitors suggests that viral proteases can be the valid molecular targets for the development of antiviral drugs against other viruses. Intensive genetic and biochemical studies have been conducted on viral proteases and new insights and results are rapidly emerging. This work reviews features of viral proteases with respective to the development of effective antiviral therapy.

In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs.

The long-term treatment of herpesvirus infections with current antivirals leads to the development of drug-resistant viruses. Because currently available antivirals finally target the viral DNA polymerase, mutant resistant to one drug often shows cross-resistance to other drugs. This evidence highlights the need for the development of new antivirals that have the different viral targets. Recently, high-through-put screening of large compound collections for inhibiting specific viral enzymes, or in vitro cell culture assay, has identified several new antivirals. These include the inhibitors of helicase/primase complex, terminase complex, portal protein and UL97 protein kinase. This review will focus on these new compounds that directly inhibit viral replication.

Background: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary., Results: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection., Conclusion: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Herpesviruses cause a wide variety of human diseases ranging from cold sores and genital herpes to encephalitis, congenital infections and lymphoproliferative diseases. These opportunistic viruses cause major problems in immunocompromised individuals such as transplant recipients, cancer patients, and HIV-infected persons. The current treatment of these infections is not optimal and there is a need for more active, less toxic compounds that might be used in place of or in addition to current therapies. We have evaluated a new series of 4-oxo-dihydroquinolines, which have a different mechanism of action than nucleosides and have activity against multiple herpesviruses. Of the four new compounds evaluated, two (PHA-529311 and PHA-570886) had greater activity than the parent, PHA-183792, against several herpesviruses and one (PHA-568561) was as effective as the parent. A fourth, PHA-243672, was considerably less effective. They had greater efficacy against cytomegalovirus (CMV) than the other herpesviruses tested and also had activity against acyclovir-resistant herpes simplex virus and varicella-zoster virus isolates and ganciclovir or foscarnet-resistant CMV isolates. These results confirm the broad-spectrum efficacy of these compounds against multiple herpesviruses and suggest that members of this class may have a potential role for treatment of a variety of herpesvirus infections.

Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus-encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.