Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis.

Meiosis 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.

Characterization of N- and C-terminal endogenously tagged Tyrosyl-DNA phosphodiesterase 2 (TDPT-1) C. elegans strains.

We have generated Tyrosyl-DNA phosphodiesterase 2 (TDPT-1) C. elegans strains where CRISPR/Cas9 was used to endogenously tag the protein at either the C- or N-terminus and validated the functionality of the resulting tagged TDPT-1 proteins. We have found that both the N-terminally tagged ( wrmScarlet::tdpt-1) and C-terminally tagged ( tdpt-1::3xflag ) worm TDPT-1 does not affect embryonic viability compared to wild type. Using the N-terminally tagged wrmScarlet::tdpt-1 strain we show, for the first time, that TDPT-1 is expressed in nuclei of the germ line and the soma. Moreover, we validate the expression of TDPT-1 at the protein level using the C-terminally tagged ( tdpt-1::3xflag ) strain.

Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans.

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

A Common Live-Attenuated Avian Herpesvirus Vaccine Expresses a Very Potent Oncogene.

Vaccines play a crucial role in the protection of animals and humans from deadly pathogens. The first vaccine that also protected against cancer was developed against the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV infects chickens and causes severe immunosuppression, neurological signs, and fatal lymphomas, a process that requires the viral oncogene, meq The most frequently used Marek's disease vaccine is the live-attenuated CVI988/Rispens (CVI) strain, which efficiently protects chickens and prevents tumorigenesis. Intriguingly, CVI expresses at least two isoforms of meq; however, it remains unknown to what extent these isoforms contribute to virus attenuation. In this study, we individually examined the contribution of the two CVI-meq isoforms to the attenuation of the vaccine. We inserted the respective isoforms into a very virulent MDV (strain RB-1B), thereby replacing its original meq gene. Surprisingly, we could demonstrate that the longer isoform of meq strongly enhanced virus-induced pathogenesis and tumorigenesis, indicating that other mutations in the CVI genome contribute to virus attenuation. On the contrary, the shorter isoform completely abrogated pathogenesis, demonstrating that changes in the meq gene can indeed play a key role in virus attenuation. Taken together, our study provides important evidence on attenuation of one of the most frequently used veterinary vaccines worldwide.IMPORTANCE Marek's disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current "gold standard" vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens.

Transcriptional Analyses of Innate and Acquired Immune Patterning Elicited by Marek's Disease Virus Vaccine Strains: Turkey Herpesvirus (HVT), Marek's Disease Virus 2 (strain SB1), and Bivalent Vaccines (HVT/SB1 and HVT-LT/SB1).

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1ß), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.

Análisis transcripcionales de patrones inmunes innatos y adquiridos inducidos por cepas vacunales del virus de la enfermedad de Marek: virus herpes del pavo (HVT), virus de Marek 2 (cepa SB1) y vacunas bivalentes (HVT/SB1 y HVT-LT/SB1). La enfermedad de Marek (MD) es una patología compleja de los pollos causada por el virus de Marek (MDV) 1 y se observa como parálisis, depresión inmune, signos neurológicos y la formación rápida de linfomas de células T. La incidencia de la enfermedad de Marek en pollos de engorde comerciales se controla en gran medida a través de la vacunación, ya sea in ovo o al momento de la eclosión con vacunas vivas atenuadas, por ejemplo, herpesvirus de pavo (HVT) o una combinación bivalente de HVT con la cepa SB1. Para ampliar aún más la protección conferida por la vacuna bivalente HVT/SB-1, los HVT recombinantes que codifican transgenes de otros virus aviares se han utilizado de forma similar para la administración in ovo. A pesar de décadas de uso, los mecanismos específicos asociados con la protección inducida por la vacuna siguen sin ser esclarecidos completamente. Además, el mecanismo para la sinergia de la vacuna conferida por la vacuna bivalente HVT/SB-1, en comparación con la administración de la cepa HVT o de la cepa SB-1 por sí solas, es en gran medida desconocida. En el presente estudio, se informa sobre los cambios temporales en la expresión genética de patrones inmunes innatos y adquiridos mediante la infección de esplenocitos ex vivo y en modelos de vacunación in ovo. Se reporta que en el modelo de infección de esplenocitos ex vivo, por 72 horas después de la infección, las vacunas indujeron IFN y la expresión de genes estimulada por IFN, con menor inducción de citocinas proinflamatorias. Para varios genes (TLR3, IFNc, OASL, Mx1, NOS2A e IL-1ß), los efectos sobre la expresión de genes fueron aditivos para la infección por HVT, SB1 y HVT/SB1. Se Observaron patrones de inducción similares en embriones de pollo y pollos de engorde comerciales vacunados in ovo con HVT/SB-1 o con la combinación bivalente recombinante basada en HVT (HVT-LT/SB-1). Además, la vacunación in ovo con HVT/SB-1 o HVT-LT/SB-1 parecen acelerar la maduración inmune, con patrones de expresión que sugieren una migración acelerada de células T y células asesinas naturales en el bazo. Finalmente, la vacuna HVT/SB-1 dio como resultado una inducción coordinada de IL-12p40 y una regulación a la baja de supresores de las señales de citocinas 1 y 3, indicativas de los patrones clásicos de macrófagos 1 y células cooperadoras tipo 1.