Epstein-Barr virus-encoded RNAs (EBERs) complement the loss of the herpesvirus telomerase RNA (vTR) in virus-induced tumor formation

Marek’s disease virus (MDV) is an alphaherpesvirus that causes fatal lymphomas in chickens and is used as a natural virus-host model for herpesvirus-induced tumorigenesis. MDV encodes a telomerase RNA subunit (vTR) that is crucial for efficient MDV-induced lymphoma formation; however, the mechanism is not completely understood. Similarly, Epstein Barr-virus (EBV) encodes two RNAs (EBER-1 and EBER-2) that are highly expressed in EBV-induced tumor cells, however their role in tumorigenesis remains unclear. Intriguingly, vTR and EBER-1 have interaction partners in common that are highly conserved in humans and chickens. Therefore, we investigated if EBER-1 and/or EBER-2 can complement the loss of vTR in MDV-induced tumor formation. We first deleted vTR (vΔvTR) and replaced it by either EBER-1 or EBER-2 in the very virulent RB-1B strain. Insertion of either EBER-1 or EBER-2 did not affect MDV replication and their expression levels were comparable to vTR in wild type virus. Intriguingly, EBER-2 restored tumor formation of MDV that lacks vTR. EBER-1 partially restored MDV oncogenicity, while tumor formation was severely impaired in chickens infected with vΔvTR. Our data provides the first evidence that EBERs possess tumor-promoting properties in vivo using this natural model for herpesvirustumorigenesis

The Transcriptional Landscape of Marek’s Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes

Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and poses a serious threat to poultry health. In infected animals, MDV efficiently replicates in B cells in various lymphoid organs. Despite many years of research, the viral transcriptome in primary target cells of MDV remained unknown. In this study, we uncovered the transcriptional landscape of the very virulent RB1B strain and the attenuated CVI988/Rispens vaccine strain in primary chicken B cells using high-throughput RNA-sequencing. Our data confirmed the expression of known genes, but also identified a novel spliced MDV gene in the unique short region of the genome. Furthermore, de novo transcriptome assembly revealed extensive splicing of viral genes resulting in coding and non-coding RNA transcripts. A novel splicing isoform of MDV UL15 could also be confirmed by mass spectrometry and RT-PCR. In addition, we could demonstrate that the associated transcriptional motifs are highly conserved and closely resembled those of the host transcriptional machinery. Taken together, our data allow a comprehensive re-annotation of the MDV genome with novel genes and splice variants that could be targeted in further research on MDV replication and tumorigenesis

In vitro infection of primary chicken lymphocytes with Marek’s disease virus

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects immune cells and causes a deadly lymphoproliferative disease in chickens. Cytokines and monoclonal antibodies promote the survival of chicken lymphocytes in vitro. Here, we describe protocols for the isolation, maintenance, and efficient MDV infection of primary chicken lymphocytes and lymphocyte cell lines. This facilitates the investigation of key aspects of the MDV life cycle in the primary target cells of viral replication, latency, genome integration, and reactivation. For complete details on the use and execution of this protocol, please refer to Schermuly et al.,1 Bertzbach et al. (2019),2 and You et al.3 For a comprehensive background on MDV, please see Osterrieder et al.4 and Bertzbach et al. (2020).5 Subject areas: Cell Biology, Cell isolation, Cell-based Assays, Microbiolog

Viral and cellular telomerase RNAs possess host-specific anti-apoptotic functions

Human telomerase RNA (hTR) is overexpressed in many cancers and protects T cells from apoptosis in a telomerase-independent manner. The most prevalent cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek’s disease virus (MDV). MDV encodes a viral telomerase RNA (vTR) that plays a crucial role in MDV-induced tumorigenesis and shares all four conserved functional domains with hTR. In this study, we assessed whether hTR drives tumor formation in this natural model of herpesvirus-induced tumorigenesis. Therefore, we replaced vTR with hTR in the genome of a highly oncogenic MDV. Furthermore, we investigated the anti-apoptotic activity of vTR, hTR, and their counterpart in the chicken [chicken telomerase RNA (cTR)]. hTR was efficiently expressed and did not alter replication of the recombinant virus. Despite its conserved structure, hTR did not complement the loss of vTR in virus-induced tumorigenesis. Strikingly, hTR did not inhibit apoptosis in chicken cells, but efficiently inhibited apoptosis in human cells. Inverse host restriction has been observed for vTR and cTR in human cells. Our data revealed that vTR, cTR, and hTR possess conserved but host-specific anti-apoptotic functions that likely contribute to MDV-induced tumorigenesis

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field

Telomeres and Telomerase: Role in Marek’s Disease Virus Pathogenesis, Integration and Tumorigenesis

Telomeres protect the ends of vertebrate chromosomes from deterioration and consist of tandem nucleotide repeats (TTAGGG)n that are associated with a number of proteins. Shortening of the telomeres occurs during genome replication, thereby limiting the replication potential of somatic cells. To counteract this shortening, vertebrates encode the telomerase complex that maintains telomere length in certain cell types via de novo addition of telomeric repeats. Several herpesviruses, including the highly oncogenic alphaherpesvirus Marek’s disease virus (MDV), harbor telomeric repeats (TMR) identical to the host telomere sequences at the ends of their linear genomes. These TMR facilitate the integration of the MDV genome into host telomeres during latency, allowing the virus to persist in the host for life. Integration into host telomeres is critical for disease and tumor induction by MDV, but also enables efficient reactivation of the integrated virus genome. In addition to the TMR, MDV also encodes a telomerase RNA subunit (vTR) that shares 88% sequence identity with the telomerase RNA in chicken (chTR). vTR is highly expressed during all stages of the virus lifecycle, enhances telomerase activity and plays an important role in MDV-induced tumor formation. This review will focus on the recent advances in understanding the role of viral TMR and vTR in MDV pathogenesis, integration and tumorigenesis

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development

Telomeres and Telomerase: Role in Marek’s Disease Virus Pathogenesis, Integration and Tumorigenesis

Telomeres protect the ends of vertebrate chromosomes from deterioration and consist of tandem nucleotide repeats (TTAGGG)n that are associated with a number of proteins. Shortening of the telomeres occurs during genome replication, thereby limiting the replication potential of somatic cells. To counteract this shortening, vertebrates encode the telomerase complex that maintains telomere length in certain cell types via de novo addition of telomeric repeats. Several herpesviruses, including the highly oncogenic alphaherpesvirus Marek’s disease virus (MDV), harbor telomeric repeats (TMR) identical to the host telomere sequences at the ends of their linear genomes. These TMR facilitate the integration of the MDV genome into host telomeres during latency, allowing the virus to persist in the host for life. Integration into host telomeres is critical for disease and tumor induction by MDV, but also enables efficient reactivation of the integrated virus genome. In addition to the TMR, MDV also encodes a telomerase RNA subunit (vTR) that shares 88% sequence identity with the telomerase RNA in chicken (chTR). vTR is highly expressed during all stages of the virus lifecycle, enhances telomerase activity and plays an important role in MDV-induced tumor formation. This review will focus on the recent advances in understanding the role of viral TMR and vTR in MDV pathogenesis, integration and tumorigenesis

Tumorfördernde Eigenschaften der zellulären Telomerase-RNA und viraler RNAs in Herpesvirus-induzierter Krebsentstehung

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. MDV encodes a viral telomerase RNA (vTR) that is highly expressed during all stages of the virus life cycle. vTR is crucial for efficient MDV-induced lymphoma formation, however, the mechanism is not completely understood. Despite the high sequence identity between vTR and the cellular telomerase RNA (chTR) of 88%, it remains elusive if the overexpression of the chTR can contribute to cellular transformation. Intriguingly, TRs and Epstein- Barr virus (EBV) encoded RNAs (EBER-1 and EBER-2) have interaction partners in common that are highly conserved in humans and chickens. EBERs are the most abundant viral transcripts in EBV- induced tumor cell. However, their role in tumor development is still controversial. In the first part of the study, we wanted to investigate if the overexpression of cellular TRs (chTR) have tumor-promoting functions using a natural virus-host animal model of herpesvirus tumorigenesis. We initially deleted vTR (vΔvTR) in the RB-1B genome, a very virulent MDV strain, and subsequently inserted chTR at the vTR locus resulting in vchTR, using the Two- step Red-mediated mutagenesis system. The expression levels of vTR and chTR (in vchTR) were confirmed using qRT-PCR. chTR expression levels in vchTR were comparable to vTR in the wild-type. Neither the vTR-deletion nor the chTR insertion effected the MDV replication properties in vitro and in vivo. Intriguingly, the tumor formation was severely impaired in the absence of vTR while, the tumor formation in the chickens infected with vchTR was similar to those infected with the wild-type or revertant virus. Our results provided the first evidence that the overexpression of the cellular TRs can complement the functions of vTR in MDV-induced tumorigenesis. In the second part of this study, we wanted to address if EBERs (EBER-1 and EBER-2) possess tumor promoting functions and can transform chicken T cells using a small animal model for MDV-tumorigenesis. We generated recombinant MDVs expressing either EBER-1 or EBER-2 instead of vTR, termed vEBER-1 and vEBER-2. Expression levels of EBERs were detected during the viral lytic replication in vitro. EBERs were highly expressed and comparable to vTR expression in the wild-type or revertant. Furthermore, the recombinant mutants were replicating efficiently in cell culture and in infected animals. To assess the tumor promoting properties of EBERs, we performed an animal experiment where the infected animals were monitored for tumor development. EBERs partially restored the tumor formation if compared to the vTR-deletion. Tumor incidence with vEBER-2 was higher than with vEBER-1 compared to the wild-type. Our results for this aim displayed the potential tumorigenicity of EBERs their ability to transform different host immune cells. Furthermore, it provided a useful model to investigate the activities of EBERs in the cellular transformation and the underlying mechanism using a small animal model for virus-induced cancer formation.Das Virus der Marek‘schen Krankheit (MDV) ist ein onkogenes Alphaherpesvirus welches zur Entstehung von tödlichen Lymphomen führt. MDV kodiert für eine virale Telomerase RNA (vTR), die in allen Stufen des MDV-Lebenszyklus stark exprimiert wird. vTR ist für die durch MDV induzierte Tumorentstehung essentiell, wobei der zugrundeliegende Mechanismus nicht komplett verstanden ist. Trotz der hohen Sequenzhomologie zwischen vTR und der Wirtszell- Telomerase RNA (chTR) von 88% ist nicht untersucht ob die Überexpression von chTR zur Zellentartung beiträgt. Interessanterweise haben TRs und Ebstein- Barr-Virus (EBV)-kodierte RNAs (EBER-1 und EBER-2) gemeinsame Wechselwirkungspartner, welche in Menschen und Hühnern hochkonserviert sind. EBERs sind die am stärksten auftretenden viralen Transkripte in EBV Tumorzellen. Die Rolle dieser EBERs in der Tumorentstehung ist jedoch strittig. Ziel des ersten Teils der Dissertation war die Untersuchung, ob eine Überexpression der zellulären TRs (chTRs) tumorbegünstigende Funktionen im Tiermodell für herpesvirusinduzierte Tumorentstehung haben. In einer vTR Deletionsmutante (vΔvTR) des hochvirulenten MDV Feldstammes RB-1B wurde durch Two-step Red-mediierte Mutagenese chTR in den vTR Locus eingesetzt (vchTR). Per qRT-PCR wurden die vTR und chTR (in vchTR) Expressionsniveaus der Virusmutanten bestimmt. chTR Expressionsniveaus in vchTR war mit vTR im Wildtyp-Virus vergleichbar. Weder die vTR-Deletion noch die chTR Insertion hatte Einfluss auf die Virusreplikation in vitro und in vivo. Die Tumorentstehung war in Abwesenheit von vTR erheblich reduziert wohingegen die Tumorinzidenz in den vchTR infizierten Hühnern vergleichbar mit der Wildtyp- und der Revertantengruppe war. Diese Daten liefern die ersten Hinweise darauf, dass die Überexpression von zellulären TRs die Funktionen von vTR in der MDV induzierten Tumorentstehung komplementieren kann. Im zweiten Teil der Dissertation haben wurde Rolle der EBERs (EBER-1 und EBER-2) adressiert. Dabei war das Ziel herauszufinden ob die EBERs tumorbegünstigende Funktionen besitzen und Hühner-T-Zellen im Tiermodell für MDV-Tumorgenese transformieren können. Hierzu wurden rekombinante MDV Mutanten generiert die entweder EBER-1 oder EBER-2 an Stelle von vTR exprimieren: vEBER-1 bzw. vEBER-2. Die Expressionsniveaus beider EBERs wurde in lytisch infizierten Zellen in vitro getestet. Beide EBERs wurden stark exprimiert und waren mit der vTR Expression vom Wildtyp und der Revertante vergleichbar. Zusätzlich wurde eine effiziente Virusreplikation in Zellkultur und im Tiermodell beobachtet. Um die tumorfördernden Eigenschaften der EBERs zu untersuchen wurde ein Tierversuch durchgeführt, wobei die Tiere auf Tumorentstehung kontrolliert wurden. Die EBERs konnten, im Vergleich zur vTR Deletionsmutante, die Tumorentstehung teilweise wiederherstellen. Die Tumorinzidenz von vEBER-2 war höher als die von vEBER-1 verglichen mit dem Wildtyp. Diese Ergebnisse zeigen erstens mögliche tumorfördernde Eigenschaften der EBERs – auch, dass diese unterschiedliche Wirts-Immunzellen transformieren können. Zweitens bieten sie ein nützliches Tiermodell für virusinduzierte Krebsentstehung um die EBER- Aktivitäten in Zelltransformationen und die hier zugrundeliegenden Mechanismen zu untersuchen