Deciphering the mechanism of the viral telomerase RNA in Marek’s disease virus pathogenesis and tumorigenesis
Final Report Abstract
Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T cell lymphoma in chickens. MDV encodes a telomerase RNA (vTR) that plays a crucial role in MDV-induced tumor formation. It shares an 88% sequence identity with the cellular TR in chicken (cTR) and promotes telomerase activity. We previously demonstrated that the tumor-promoting functions of vTR are independent of its role in the telomerase complex. Furthermore, we showed that vTR interacts with and relocalizes the cellular protein RpL22, which is an important factor in T cell development and transformation. Until now, the role of vTR-RpL22 interaction in MDV-induced tumor formation remains elusive. In addition, our preliminary data revealed that vTR has additional interaction partners and possesses anti-apoptotic functions. Considering these aspects, we hypothesized that the interaction of vTR with RpL22 and/or other interaction partners as well as vTR-mediated inhibition of apoptosis contribute to tumor formation. We addressed this hypothesis in three specific aims. Specifically, we aimed to 1) decipher the role of the vTR-RpL22 interaction in MDV- induced tumorigenesis, 2) identify novel vTR interaction partners and determine if they contribute to tumor cell proliferation 3) investigate the mechanism that allows vTR to inhibit apoptosis. To achieve aim 1, we set to abrogate the vTR-RpL22 interaction through mutating the RpL22 consensus binding sites in vTR. We identified the RpL22 binding sites in vTR and we mutated the stem loops harbor these consensus motifs. To determine if these mutations abrogate the vTR-RpL22 interaction, we performed RNA-IP assays [1]. Our RNA-IP assays revealed that, vTR variants with mutated RpL22 binding sites are still able interact to RpL22, suggesting that additional mutations of vTR stem loops are required to abrogated the interactions, which und unfortunately would alter the structure of vTR. As a contingency plan, we set to knockout RpL22 expression using CRISPR/Cas9 system in virus infected cells. Therefore, we generated recombinant MDV (rMDV) expressing Cas9 and RpL22 specific gRNAs. Even though the recombinant virus replicated efficiently, the virus rapidly lost its Cas9 expression over time, likely due to a negative selection pressure. As an alternative, we set to knockdown RpL22 expression in virus-infected cells using the shRNA technology. shRNAs against RpL22 efficient knockdown RpL22 expression. To investigate the role of RpL22 in MDV-induced tumorigenesis, we inserted the shRNA cassette into MDV genome. The rMDV efficiently replicated in culture and the shRNAs cassette was stably expressed in virus-infected cells. In addition, we inserted an RpL22 expression cassette into the MDV genome to investigate if overexpression of RpL22 alters MDV- induced tumorigenesis. The role of RpL22 in MDV-induced tumorigenesis will be investigated in vivo in the coming months. To address aim 2, to identify new vTR-new interactions, we performed several RNA- IP assays using the biotin labeled vTR-RNA. The biotin-labeled RNAs were incubated with the total chicken fibroblasts or T cells lysates. vTR stem-loop mutants were also included. Several distinct protein bands were observed on coomassie gels that likely represent vTR interaction partners. The analysis of the vTR-interactions is still ongoing at the recently established Omic unit at the Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin. Once the analyses are performed, we will identify the novel interactions and will investigate their role in the proliferation of the tumor cells. To address aim 3, in the preliminary data of this project, we demonstrate that vTR can inhibit apoptosis, a property that also has been recently reported for human TR (hTR) [2]. Our analysis showed that vTR inhibits apoptosis in different chicken cells like ESCDL and T cells. We also demonstrated that the vTR-mediated anti-apoptotic activity is independent of its role in the telomerase complex as vTR P6.1mutant that is not incorporated into the complex can efficiently inhibit apoptosis in chicken T cells. Further, we reported that the overexpression of chicken homologue cTR can inhibit apoptosis, indicating that vTR-mediated antiapoptotic activity has been acquired from the host during virus evolution. As TRs possess a conserved structure, we assessed if human TR (hTR) can complement the loss of vTR in the MDV genome. Surprisingly, hTR did not promote tumor formation in chickens. To understand why hTR does not promote MDV-induced tumorigenesis, we determined the ability of hTR and vTR to inhibit apoptosis in human and chicken cells. We discovered that hTR can efficiently inhibit apoptosis in human cells but not chicken cells. We showed here for the first time that, vTR and cellular TRs of humans and chickens possess host-specific antiapoptotic functions. To investigate which apoptosis pathway is inhibited by vTR, we induced apoptosis in chicken T cells using intrinsic and extrinsic stimuli and analyzed the pathway using Caspase-Glo assays. Our analysis revealed that both extrinsic and intrinsic pathways of apoptosis are activated in T cells with the use of intrinsic stimuli. Alternative to caspase activity, we currently assessing the mitochondrial membrane potential and the cytochrome C release upon the induction of intrinsic stimuli in T cells. Furthermore, we are currently set to analyze the expression of pro- or anti-apoptotic factors in T cell lines stably transfected with vTR using single cell RNA-seq assays. Other vTR mutants such as P6.1or P6/P8 stem-loop mutants as well as cTR and hTR are considered as well. Taken together, we demonstrated the TRs-mediated anti-apoptotic functions are hostspecific and we reported that vTR-mediated anti-apoptoic activity is independent of its role in the telomerase complex. Further, we developed an approach that facilitates the knockdown of the host factors and address their role in MDV-induced pathogenesis and tumorigenesis. Until now, in the context of this DFG grant KA 3492/8-1, we successfully published one high impact publication. The results on the different aims will be ready for publication in the next months. The delay is the project is mostly due to the Corona pandemic with its lockdowns/shutdowns, however we are confident that we will complete all proposed work in the coming months. The Results on the different (sub) aims will be highlighted in the following section.
Publications
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Viral and cellular telomerase RNAs possess host-specific anti-apoptotic functions. Microbiology Spectrum, 11(5).
Kheimar, Ahmed; Trapp-Fragnet, Laetitia; Conradie, Andelé M.; Bertzbach, Luca D.; You, Yu; Sabsabi, Mohammad A. & Kaufer, Benedikt B.
