The current status and future directions of myxoma virus, a master in immune evasion

Myxoma virus (MYXV) gained importance throughout the twentieth century because of the use of the highly virulent Standard Laboratory Strain (SLS) by the Australian government in the attempt to control the feral Australian population of Oryctolagus cuniculus (European rabbit) and the subsequent illegal release of MYXV in Europe. In the European rabbit, MYXV causes a disease with an exceedingly high mortality rate, named myxomatosis, which is passively transmitted by biting arthropod vectors. MYXV still has a great impact on European rabbit populations around the world. In contrast, only a single cutaneous lesion, restricted to the point of inoculation, is seen in its natural long-term host, the South-American Sylvilagus brasiliensis and the North-American S. Bachmani. Apart from being detrimental for European rabbits, however, MYXV has also become of interest in human medicine in the last two decades for two reasons. Firstly, due to the strong immune suppressing effects of certain MYXV proteins, several secreted virus-encoded immunomodulators (e.g. Serp-1) are being developed to treat systemic inflammatory syndromes such as cardiovascular disease in humans. Secondly, due to the inherent ability of MYXV to infect a broad spectrum of human cancer cells, the live virus is also being developed as an oncolytic virotherapeutic to treat human cancer. In this review, an update will be given on the current status of MYXV in rabbits as well as its potential in human medicine in the twenty-first century

The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells

Neue Erkenntnisse zur Rolle des Glykoproteins B und pUS3 während der Pathogenese des equine Herpesviruses

Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry revealing no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. The significant attenuation of virus growth in the case of EHV-4_gB1 may be attributable to the loss of functional interaction between gB and other proteins involved in virus entry. One possible cause for the loss of function/interaction may be structural differences between gB1 and gB4. Our rationale was that this structural difference could be caused by the different locations of the furin cleavage site within the respective gBs. To investigate the contribution of furin- mediated gB cleavage to EHV-1 and EHV-4 growth, the cleavage sites were mutated. While mitigating furin recognition motif did not affect in vitro growth of EHV-1, reconstitution of the mutant EHV-4 was not successful, which confirms previous results indicating different properties of gB4 when compared to gB1. Western blot and mass spectrometry analysis of mutated gB1 suggest that mutating the furin cleavage site indeed prevented gB cleavage and resulted in a partial misfolding. In addition, a novel signal peptide cleavage site was identified for gB1 between residues 98 and 99, which is different from that previously published. We conclude that furin cleavage is solely responsible for gB cleavage and involved in the protein folding. After addressing the structural and functional aspects of gB on a protein level, we looked at how this translated to the function of gB during a key step of EHV-1 pathogenesis, namely the viral transfer between infected PBMC to EC. Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. Infection assays revealed that EHV-1 infected B-lymphocytes and monocytes more efficiently than EHV-4. In order to evaluate viral transfer between infected PBMC and EC, co-cultivation assays were performed. Only EHV-1 was transferred from PBMC to EC and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taken together, we conclude that systemic spread and EC infection of EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer.Das Glykoprotein B (gB) spielt zusammen mit gD und dem gH/gL-Komplex eine wichtige Rolle während des Eintritts der Alphaherpesviren. Um eventuelle funktionale Unterschiede zwischen den Glykoproteinen gB1 von EHV-1 und gB4 von EHV-4 zu untersuchen, wurden die entsprechenden Gene zwischen EHV-1 und EHV-4 ausgetauscht. Das rekombinante, gB4 enthaltende EHV-1 Virus (EHV-1_gB4) wurde hinsichtlich seines Wachstums in vitro, seines Zelltropismus und des Zelleintritts untersucht, wobei keine signifikanten Unterschiede im Vergleich zum Ursprungsvirus festgestellt werden konnten. Zusätzlich wurde ein potentielles Integrin-Bindemotiv mutiert, was jedoch keinen Einfluss auf die in vitro Funktion von gB hatte. Im Gegensatz dazu konnte eine signifikante Reduzierung der Plaquegröße und der Virusvermehrung des gB1 enthaltendem EHV-4 Viruses (EHV-4_gB1) im Vergleich zum Ursprungsvirus und der Revertante ermittelt werden. Die Abnahme der Virusvermehrung könnte hierbei auf den Verlust der Interaktion von gB mit anderen Glykoproteinen, einschließlich gD und gH/gL, zurückzuführen sein, die am Zelleintritt beteiligt sind. Alternativ dazu könnte gB4 eine zusätzliche Funktion ausüben, die für die Vermehrung von EHV-4 von Nöten ist, jedoch nicht von gB1 übernommen werden kann. Die verringerte Vermehrungsfähigkeit von EHV-4_gB1 könnte auf einen Verlust der Interaktionsfähigkeit von gB mit anderen, am Zelleintritt beteiligten Proteinen zurückzuführen sein, wobei dies auf strukturellen Unterschieden von gB1 und gB4 beruhen könnte. Unsere Erklärung für einen strukturellen Unterschied ist, dass dieser durch eine unterschiedliche Lage der Furinschnittstelle in den entsprechenden gBs hervorgerufen werden könnte. Um den Einfluss der durch Furin vermittelten Spaltung von gB auf das Viruswachstum von EHV-1 und EHV-4 zu untersuchen, wurden die Schnittstellen mutiert. Während die Veränderung der Furin-Erkennungssequenz das Wachstum von EHV-1 nicht beeinflusste, konnte EHV-4 nicht rekonstituiert werden, was die oben beschrieben Ergebnisse bezüglich unterschiedlicher Eigenschaften von gB4 im Vergleich zu EHV-1 bestätigt. Obwohl die Vermehrung von EHV-1 nicht beeinflusst war, konnten Westernblot und massenspektrometrische Analysen zeigen, dass die Mutation der Furin-Erkennungssequenz tatsächlich die Spaltung von gB verhinderte und das Protein partiell fehlerhaft gefaltet war. Darüber hinaus konnte eine neue, bisher nicht publizierte Peptidschnittstelle zwischen den Aminosäuren 98TS99 identifiziert werden. Daraus schließen wir, dass allein Furin für die Spaltung von gB verantwortlich ist und somit wichtig für die korrekte Faltung des Proteins. Nachdem die strukturellen und funktionalen Aspekte von gB auf Proteinebene untersucht wurden, haben wir die Frage gestellt, wie sich die Funktion von gB auf eine der Schlüsselstellen der EHV-1 Pathogenese, nämlich der Übertagung der Viren von infizierten PBMCs (Peripheral Blood Mononuclear Cells oder mononukleäre Zellen des peripheren Blutes) auf Endothelzellen (EZ), überträgt. Infizierte PBMCs transportieren hoch effizient EHV-1, aber nicht EHV-4, zu den Endothelzellen, welche die Blutgefäße des schwangeren Uterus und des zentralen Nervensystems auskleiden, wodurch es zu Fehlgeburten bzw. Myeloenzephalopathie komme kann. Mittels eines dynamischen in vitro Modelles haben wir die Unterschiede von EHV-1 und EHV-4 hinsichtlich der Infektion von PBMCs und der PBMC/EZ-Interaktion untersucht. Infektionsversuche zeigten dabei, dass EHV-1 B-Lymphozyten und Monozyten besser infiziert als EHV-4. Um nun den Virustransfer von infizierten PBMCs auf Endothelzellen zu untersuchen, wurden Ko-Kultivierungsversuche durchgeführt. Ausschließlich EHV-1 wurde in diesem System von PBMCs auf Endothelzellen übertragen, wobei hauptsächlich gB für diesen Zell-zu-Zell-Transfer verantwortlich war. Um die dynamischen Aspekte der Interaction von PBMCs und Endothelzellen zu beleuchten, wurden infizierte PBMCs unter Anwesenheit von neutralisierenden Antiköpern durch einen mit Endothelzellen bewachsenen Durchflusskanal gepumpt. Dadurch ist es möglich, den kapillaren Blutstrom zu simulieren und währenddessen das Verhalten der infizierten PBMCs durch „live cell imaging“ und automatischer Zell-Verfolgung (cell tracking) zu analysieren. Hierbei ließ sich feststellen, dass EHV-1 in der Lage war, das Anhaften und Rollen der infizierten PBMCs auf den Endothelzellen effizienter aufrechtzuerhalten als EHV-4. Zusammengefasst lässt sich daraus folgern, dass die systemische Ausbreitung und Infektion von Endothelzellen durch EHV-1, aber nicht EHV-4, durch die Fähigkeit vermittelt wird, mononukleäre Blutzellen zu infizieren und/oder sie hinsichtlich ihres Anhaftungs- und Rollverhaltens auf Endothelzellen und der anschließenden Infektion umzuprogrammieren

Comparative Analysis of Glycoprotein B (gB) of Equine Herpesvirus Type 1 and Type 4 (EHV-1 and EHV-4) in Cellular Tropism and Cell-to-Cell Transmission

Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry rivaling no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. In conclusion, our results show that the exchange of gB between EHV-1 and EHV-4 is possible, but results in a significant attenuation of virus growth in the case of EHV-4_gB1. The generation of stable recombinant viruses is a valuable tool to address viral entry in a comparative fashion and investigate this aspect of virus replication further

Combining Oncolytic Viruses and Small Molecule Therapeutics: Mutual Benefits

The focus of treating cancer with oncolytic viruses (OVs) has increasingly shifted towards achieving efficacy through the induction and augmentation of an antitumor immune response. However, innate antiviral responses can limit the activity of many OVs within the tumor and several immunosuppressive factors can hamper any subsequent antitumor immune responses. In recent decades, numerous small molecule compounds that either inhibit the immunosuppressive features of tumor cells or antagonize antiviral immunity have been developed and tested for. Here we comprehensively review small molecule compounds that can achieve therapeutic synergy with OVs. We also elaborate on the mechanisms by which these treatments elicit anti-tumor effects as monotherapies and how these complement OV treatment

The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells