Browsing by Author "Bhandari, Nirajan"
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Item Effects of Meq mutations on Marek's disease virus (MDV): pathogenecity and oncogenicity(University of Delaware, 2017) Bhandari, NirajanMarek’s disease (MD) is a highly transmissible lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek’s disease virus-1 (MDV-1). MD remains a constant problem to poultry production worldwide, due to the cost of vaccination and the continuous evolution of more virulent field strains. Upon analyzing MDV-1 strains of distinct pathotypes, we, and others, have found that the major oncoprotein (Meq) of MDV-1 strains has specific coding mutations that correlate with virulence level. We, and others, have previously reported differences in the transcriptional activation potential and the cellular binding profiles of the different Meq isoforms. We therefore hypothesized that these mutations could directly affect the virulence of MDV-1 strains. Using RB-1B as a well characterized genetic backbone, we constructed recombinant MDVs that harbor the meq genes of the vaccine strain CVI988 (399 aa form), and the very virulent plus N strain (339 aa, vv+ form). Contrary to our hypothesis, pathogenicity studies in SPF chickens revealed that there was no effect of the replacement of the meq genes on MD, mortality or tumor incidence. These data therefore suggest that in unvaccinated, maternal antibody-free chickens, changes in the Meq coding sequence conferred no inherent pathogenicity increase (or decrease) for the mutations examined. Experimental validation via Southern blotting showed that both copies of meq were restored in all the recombinant viruses upon replication in CEF in vitro, SPC in vivo or in established cell lines. Similarly, PCR and sequencing of the meq loci and sequencing of meq loci ensured that viruses had not undergone significant mutations (duplications, deletions, etc.) during propagation in cell culture or in vivo, or had not been mixed during the study. ☐ A follow up hypothesis we formulated based on the aforementioned observations was that the mutations in Meq have occurred upon selection of viruses that have the ability to overcome the innate immune responses and signaling elicited from vaccination. Vaccines are presumed to be the main drivers of MDV evolution towards higher virulence. Since, the vaccines do not elicit sterilizing immunity, but only prevent MDV-induced lymphoma formation, this provides an opportunity to the virus to continually evolve in the presence of selective pressure exerted by vaccinal immunity on the full length Meq form. To address this hypothesis, we simulated innate immune activation analogous to early innate immune responses post-vaccination, using various innate immune agonists (LPS, Poly I:C, cGAMP). Replication of the recombinant viruses in CEF showed that cGAMP treatment (inducer of STING and type I interferon expression) caused a decrease in plaque number and plaque area size for RB-1B CVI Meq, but did not affect the RB-1B parent or RB-1B N Meq viruses, suggesting that Meq from mildly virulent MDV CVI988 lacks the ability to overcome cGAS-STING anti-viral signaling. On the other hand, vv and vv+ Meq forms displayed an ability to overcome cGAS-STING anti-viral signaling. Plaque number and plaque area analysis for the RB-1B N meq recombinant showed that its replication was least affected by any of the agonist treatments in both CEF and spleen cells (SPC). Thus, the N strain-meq, seems to have overcome innate signaling triggered via either of TLR3, TLR4 or cGAS-STING pathways. Agonist-treated, virus-infected SPC co-cultured with CEF ex vivo, showed a profound reduction in plaque number in the order: RB-1B CVI meq virus (~30% for each treatment) > RB-1B parent virus (~10% for each treatment) > RB-1B N meq virus (~4% for each treatment). ☐ Our work therefore suggests that mutations in the Meq oncoprotein are not directly responsible for the observed differences in MDV virulence levels, but that these Meq mutations have likely been selected indirectly based upon the pressure exerted by the innate immune responses to vaccines and it is through this evasion that they have a role in the increased MDV virulence.Item Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis(Journal of Biological Chemistry, 2024-06-28) Kwah, Ji Kent; Bhandari, Nirajan; Rourke, Christine; Gassaway, Gabriella; Jaramillo-Lambert, AimeeMeiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5′-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.Item The role of 5'-tyrosyl-DNA-phosphodiesterase two (TDPT-1) mediated suppression of DNA topoisomerase 2 (TOP-2) during meiosis in C. elegans(University of Delaware, 2023) Bhandari, NirajanMeiosis, a specialized cell division involving a single cycle of DNA replication followed by two cycles of chromosome segregation, is a highly coordinated event. Chromosome segregation errors during meiosis have hazardous consequences such as birth defects, genetic diseases, infertility and malignancy. DNA Topoisomerase II (Topo II) is required to relieve the topological stress associated with the unwinding of DNA during replication, recombination and sister chromatid segregation. In addition, Topo II has been also found to maintain chromosome structure. The focus of Topo II studies has been on its roles during mitosis but its role in meiosis is not very well defined. Our lab previously characterized an allele of top-2 [top-2(it7)] in Caenorhabditis elegans with a male meiosis-specific phenotype. top-2(it7) is a temperature sensitive (ts) allele and top-2(it7ts) sperm that develop at the restrictive temperature of 24°C have chromosome segregation defects at anaphase I of meiosis, which results in embryonic lethality after fertilization. ☐ In chapter 2, employing EMS mutagenesis followed by a genetic suppressor screen, I identified putative genes that interact with top-2 during meiosis. I used a combination of RNAi and CRISPR/Cas9 genome editing to validate the responsible suppressor mutations identified by whole-genome sequencing. I also examined the meiotic chromosome segregation in the suppressors and found that chromosome segregation defects are ameliorated in the suppressors. Interestingly, seven of the 11 identified suppressors that were identified had point mutations in the same gene, 5'-tyrosyl-DNA phosphodiesterase two (TDP2, C. elegans tdpt-1). Human TDP2 is involved in the removal of trapped TOP2-DNA protein complexes (TOP2-DPCs). ☐ tdpt-1 mutation-mediated suppression of the top-2 mutant phenotype poses an important question: What is the specific role of TDPT-1 in relation to TOP-2 in meiosis and how do these proteins interplay to help in accurate chromosome segregation during male meiosis? In chapter 3, I utilized genetic analysis to elucidate the role of these tdpt-1 mutations in context of top-2(it7) during meiosis. I found that tdpt-1 mutant mediated suppression is due to loss of function of the protein and also that the tdpt-1 mutations do not have a phenotype independently of top-2(it7) in meiosis. In addition, I found that fewer double stranded breaks (DSBs) are generated in the top-2(it7) mutant at 24°C but a tdpt-1 suppressor mutation restores DSBs levels. Moreover, I characterized and validated the functionality of endogenous fluorescent tag at N-terminus of TDPT-1 protein and utilized this tool to show that TDPT-1 is expressed in nuclei of the germ line and the soma of C. elegans. Chapter 4 is the extension of chapter 3 where I utilized an E. coli system to purify and biochemically characterize the properties of the TDPT-1 mutant proteins in relation to wild type. I showed that the suppressing mutations in TDPT-1 impair the phosphodiesterase activity and affect the thermal stability of TDPT-1. ☐ Together, the results from my dissertation work suggest the regulation of meiotic chromosomes through an interplay of two special enzymes whose function in meiosis have not been well defined previously. This dissertation has laid a foundation for more work required to further illuminate how exactly these enzymes function for accurate meiotic progression.