Sascha Trapp

Marek's disease virus: from miasma to model

Mareks disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.

The Protein Encoded by the US3 Orthologue of Marek's Disease Virus Is Required for Efficient De-Envelopment of Perinuclear Virions and Involved in Actin Stress Fiber Breakdown

ABSTRACT Mareks disease virus (MDV) encodes a protein exhibiting high amino acid similarity to the US3 protein of herpes simplex virus type 1 and the gene 66 product of varicella-zoster virus. The MDV US3 orthologue was replaced with a kanamycin resistance gene in the infectious bacterial artificial chromosome clone BAC20. After transfection of US3-negative BAC20 DNA (20ΔUS3), the resulting recombinant 20ΔUS3 virus exhibited markedly reduced growth kinetics. Virus titers on chicken embryo cells were reduced by approximately 10-fold, and plaque sizes were significantly smaller (65% reduction) compared to parental BAC20 virus. The defect of the US3-negative MDV was completely restored in a revertant virus (20US3*) expressing a US3 protein with a carboxy-terminal FLAG tag. Electron microscopical studies revealed that the defect of the 20ΔUS3 mutant to efficiently spread from cell to cell was concomitant with an accumulation in the perinuclear space of primarily enveloped virions in characteristic vesicles containing several virus particles, which resulted in reduced numbers of particles in the cytoplasm. The formation of these vesicles was not observed in cells infected with either parental BAC20 virus or the 20US3* revertant virus. The role of the MDV US3 protein in actin stress fiber breakdown was investigated by visualizing actin with phalloidin-Alexa 488 after infection or transfection of a US3 expression plasmid. Addition of the actin-depolymerizing drug cytochalasin D to cells transfected or infected with BAC20 resulted in complete inhibition of plaque formation with as little as 50 nM of the drug, while concentrations of nocodazole as high as 50 μM only had a relatively minor effect on MDV plaque formation. The results indicated that the MDV US3 serine-threonine protein kinase is transiently involved in MDV-mediated stress fiber breakdown and that polymerization of actin, but not microtubules, plays an important role in MDV cell-to-cell spread.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Mareks disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR − viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR − viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin αv. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus–host infection model.

Marek's disease virus: lytic replication, oncogenesis and control.

Marek’s disease (MD) is caused by a ubiquitous, lymphotropic alphaherpesvirus, MD virus (MDV). MD has been a major concern in the poultry industry owing to the emergence of increasingly virulent strains over the last few decades that were isolated in the face of comprehensive vaccination. The disease is characterized by a variety of clinical signs; among them are neurological symptoms, chronic wasting and, most notably, the development of multiple lymphomas that manifest as solid tumors in the viscera and musculature. Much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among the many genes encoded by MDV, a number have been shown recently to affect the development of tumors in chickens, one protein directly causing transformation of cells (Meq) and another being involved in maintaining transformed cells (vTR). Other MDV gene products modulate and are involved in early lytic in vivo replication, thereby increasing the chance of transformation occurring. In this review, we will summarize specific genes encoded by MDV that are involved in the initiation and/or maintenance of transformation and will focus mostly on current vaccination and control strategies against MD, particularly how modern molecular biological methods may be used to improve strategies to combat the disease in the future.

vLIP, a Viral Lipase Homologue, Is a Virulence Factor of Marek's Disease Virus

ABSTRACT The genome of Mareks disease virus (MDV) has been predicted to encode a secreted glycoprotein, vLIP, which bears significant homology to the α/β hydrolase fold of pancreatic lipases. Here it is demonstrated that MDV vLIP mRNA is produced via splicing and that vLIP is a late gene, due to its sensitivity to inhibition of DNA replication. While vLIP was found to conserve several residues essential to hydrolase activity, an unfavorable asparagine substitution is present at the lipase catalytic triad acid position. Consistent with structural predictions, purified recombinant vLIP did not show detectable activity on traditional phospholipid or triacylglyceride substrates. Two different vLIP mutant viruses, one bearing a 173-amino-acid deletion in the lipase homologous domain, the other having an alanine point mutant at the serine nucleophile position, caused a significantly lower incidence of Mareks disease in chickens and resulted in enhanced survival relative to two independently produced vLIP revertants or parental virus. These data provide the first evidence that vLIP enhances the replication and pathogenic potential of MDV. Furthermore, while vLIP may not serve as a traditional lipase enzyme, the data indicate that the serine nucleophile position is nonetheless essential in vivo for the viral functions of vLIP. Therefore, it is suggested that this particular example of lipase homology may represent the repurposing of an α/β hydrolase fold toward a nonenzymatic role, possibly in lipid bonding.

Marek's disease virus microRNA designated Mdv1-pre-miR-M4 targets both cellular and viral genes.

Mdv1-miR-M4 is one of 25 microRNAs (miRNAs) expressed by Marek’s disease virus (MDV-1), an oncogenic alphaherpesvirus that induces fatal T-cell lymphoma in chickens. Mdv1-miR-M4 was shown to be the second functional viral ortholog of miR-155, a cellular miRNA that plays a crucial role in several physiological and pathological processes in lymphocyte biology. In this study, we investigated a panel of putative mdv1-miR-M4 targets involved in gene networks affecting both cellular and viral life cycles. Using luciferase reporter assays, we showed that mdv1-miR-M4-5P and miR-155 efficiently targeted a common set of 3′ untranslated regions (3′UTR) of six cellular genes (GPM6B, RREB1, c-Myb, MAP3K7IP2, PU.1 and C/EBP). In addition, we also investigated the interactions between mdv1-miR-M4-5P and mdv1-miR-M43P and viral mRNAs encoding UL28 and UL32 in both reporter and western blot assays. Mdv1-miR-M4 specifically inhibited the translation of these two viral proteins, which are involved in the cleavage/packaging of herpesvirus DNA.

Potential of Equine Herpesvirus 1 as a Vector for Immunization

ABSTRACT Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.

Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Mareks disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.

Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Mareks disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity.

Immunization and challenge experiments with a new modified live bovine herpesvirus type 1 marker vaccine prototype adjuvanted with a co-polymer

Western European control programs against bovine herpesvirus type 1 (BoHV-1) infections utilize attenuated BoHV-1 marker vaccines with a deletion of the glycoprotein E (gE) encoding gene. However, a recent study demonstrated the potential risk of virulence recovery of gE-deleted BoHV-1 marker vaccine strains due to recombination (Muylkens et al. [15]). Based on an infectious bacterial artificial chromosome clone, a gE- and thymidine kinase (TK)-gene-deleted BoHV-1 mutant (BoHV-1DeltagEDeltaTK) was constructed. The recombinant virus was subsequently tested as a novel modified live marker vaccine candidate in an immunization-challenge trial using BoHV-1 seronegative calves. Additionally, a non-virucidal co-polymer was tested together with the recombinant virus acting as a vaccine-adjuvant. Animals were vaccinated twice through intramuscular injection and challenged intranasally 3 weeks later with a virulent BoHV-1 field strain. Duration and titres of challenge virus shedding were significantly reduced in all vaccinees. Importantly, reduction of challenge virus shedding and serological antibody levels in response to vaccination with vaccine preparations containing the co-polymer-adjuvant were markedly improved when compared to vaccine formulations without an adjuvant. Taken together, our study describes a novel double deletion mutant as a safe and efficacious BoHV-1-prototype marker vaccine strain with enhanced protective capacity especially when administered together with a co-polymer adjuvant.