Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase family.

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.

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase.

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.

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.

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 ß-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.

Viral Ubiquitin and Ubiquitin-Like Deconjugases-Swiss Army Knives for Infection.

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.

Terminase Large Subunit Provides a New Drug Target for Herpesvirus Treatment.

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.

Rapid and sensitive detection of elephant endotheliotropic herpesvirus 1 (EEHV1) in blood by loop-mediated isothermal amplification (LAMP).

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 101.2 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.

Thymidine kinase and protein kinase in drug-resistant herpesviruses: Heads of a Lernaean Hydra.

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 ß-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.

Broad anti-herpesviral activity of α-hydroxytropolones.

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 (pUL15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pUL15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pUL15C 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-pUL15C 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-pUL15C 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.

Herpesvirus deconjugases inhibit the IFN response by promoting TRIM25 autoubiquitination and functional inactivation of the RIG-I signalosome.

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.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

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.

Assemblins as maturational proteases in herpesviruses.

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.

Profile of anti-herpetic action of ASP2151 (amenamevir) as a helicase-primase inhibitor.

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 (EC50), 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 EC50 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.

Common Evolutionary Origin of Procapsid Proteases, Phage Tail Tubes, and Tubes of Bacterial Type VI Secretion Systems.

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.

Prevalence and Clinical Significance of Herpesvirus Infection in Populations of Australian Marsupials.

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.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

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.

Anti-herpesvirus agents: a patent and literature review (2003 to present).

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.

Ovine herpesvirus 1 (OVHV-1) thymidine kinase locus sequence analysis: evidence that OVHV-1 belongs to the Macavirus genus of the Gammaherpesvirinae subfamily.

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.

Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

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.

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

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.

Potential of protein kinase inhibitors for treating herpesvirus-associated disease.

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.