Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA’s acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.info:eu-repo/semantics/publishedVersio

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-A crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.info:eu-repo/semantics/publishedVersio

Genetic analysis of the role of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) in B cell transformation.

Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis and is associated with several human malignancies. In vitro, EBV induces the activation and continuous proliferation of primary human B cells, which carry the viral genome as a latent episome. One of the latency-associated proteins is the EBV nuclear antigen leader protein (EBNA-LP). EBNA-LP is strongly expressed at the initiation of the transformation process and has been previously reported to assist the activation of genes by EBNA-2. To investigate the role and mechanism of action of EBNA-LP in the context of viral infection and B cell transformation, I have generated EBNA-LP knockout EBVs (LP-KO i) and their revertants (LP-REV i) by recombineering in the B95.8 bacterial artificial chromosome (BAC). We found that B cells infected with LP-KO i were able to undergo limited proliferation. However, we were not able to establish LCLs after infection of EBV-negative B cells. We also found that LP-REV i was somewhat impaired in its transforming ability. We identified four nucleotide changes in EBNA-LP’s introns in both LP-KO i and LP-REV i that may influence transforming abilities. Therefore, new EBNA-LP knockouts (LP-KO w) containing wild-type intronic sequences were constructed. LP-KO w initially induced proliferation of adult B cells but then slowed around 5-14 days post infection, after which the cells recovered and established LCLs. However, in cord blood, LP-KO w failed to establish LCLs. Transcript level analysis in primary B cell infections showed that EBNA-LP can regulate the EBNA-2-regulated viral genes LMP-1 and LMP-2, as well as the EBER-2 gene, which is EBNA-2-independent. EBNA-LP has also a more limited role in modulating the EBNA-2-targeted host gene HES-1.Open Acces

The Use of Natural Sorbents to Reduce Ammonia Emissions from Cattle Faeces

Intensification of animal production leads to an increase in ammonia emissions into the environment. For this reason, various methods and strategies are sought to reduce ammonia emissions from faeces. The aim of the study was to test the possibility of using natural sorbents and sorbent mixtures to reduce ammonia emissions from cattle faeces. Faecal samples for analysis were collected from Holstein-Friesian dairy cows during the winter. The amount of ammonia emissions from cow faeces was determined every seven days, after mixing the faeces with a mixture of selected sorbents. All of the sorbents used have the potential to remove ammonia. The most effective reduction in ammonia was achieved using biochar and a mixture of bentonite with zeolite. The reduction in these groups was 42.56% and 24.56%, respectively, relative to the control group. The results indicate that these sorbents can be used to reduce ammonia emissions from cattle farms

Mimikra molekularna na poziomie epitopów rozpoznawanych przez limfocyty T w inicjacji odpowiedzi autoimmunologicznej w toczniu rumieniowatym układowym.

Toczeń rumieniowaty układowy jest złożoną chorobą autoimmunologiczną dotykającą wiele tkanek i narządów, takich jak skóra, nerki, płuca, serce. Charakterystyczną cechą tocznia jest obecność przeciwciał skierowanych przeciwko białkom własnego organizmu. Depozycja autoprzeciwciał w nerkach często prowadzi do ich uszkodzenia, co stanowi główną komplikację w tej chorobie. Aktywacja autoreaktywnych limfocytów T i ich interakcja z limfocytami B jest podstawą w generowaniu autoprzeciwciał. Nasze badania sugerują, iż molekularna mimikra pomiędzy peptydami pochodzącymi z ludzkiej mikroflory i własnymi antygenami, zaangażowana jest w aktywację autoreaktywnych limfocytów T w toczniu. Aby zweryfikować naszą hipotezę, wybraliśmy białko Ro60 jako modelowy antygen i myszy transgeniczne HLA-DR3 jako model zwierzęcy. Autoprzeciwciała skierowane przeciwko Ro60 są głównym komponentem repertuaru autoprzeciwciał w toczniu, natomiast HLA-DR3 to najwyższy genetyczny czynnik ryzyka predysponujący do tej choroby. Wykorzystując informacje na temat trzech epitopów obecnych na Ro60 i kluczowych dla aktywacji aminokwasów w tych epitopach, stworzyliśmy wzorzec, na podstawie którego przeprowadzone zostały poszukiwania peptydów stanowiących mimikrę. Peptydy te zostały zsyntetyzowane, a następnie przetestowane w kierunku ich zdolności do aktywacji hybryd limfocytów T skierowanych przeciwko Ro60. Peptydy będące w stanie zaktywować hybrydy zostały użyte do immunizacji myszy transgenicznych HLA-DR3 w celu analizy odpowiedzi autoimmunologicznej.Zidentyfikowaliśmy tysiące peptydów wykazujących mimikrę do Ro60. Duża część peptydów pochodzących z mikroorganizmów naturalnie występujących w jamie ustnej, jelitach, skórze człowieka była zdolna aktywować limfocyty T skierowane przeciwko Ro60. Odkryliśmy także, że jeden z peptydów wykazujących mimikrę do Ro60 jest w stanie zaindukować odpowiedź immunologiczną przeciwko białku La (zasocjowanemu z białkiem Ro60) w myszach transgenicznych HLA-DR3. Obecnie testujemy zdolność aktywacji hybryd limfocytów T i indukcji odpowiedzi immunologicznej przeciwko Ro60 i La przez całe białko, z którego pochodzi peptyd wykazujący mimikrę.Nasze badania sugerują, iż lifmocyty T skierowane przeciwko Ro60 mogą być aktywowane przez ekspozycję na peptydy pochodzące z różnych organizmów obecnych w ludzkiej florze bakteryjnej. Interakcja tych limfocytów T z autoreaktywnymi limfocytami B może zaindukować produkcje autoprzeciwciał. Przypuszczamy zatem, iż u pacjentów z toczniem, powtórzona ekspozycja na różne mikroogranizmy przez dłuższy czas może zainicjować odpowiedź autoimmunologiczną.Systemic lupus erythematosus (SLE) is a complex autoimmune disorder affecting multiple organs such as skin, kidneys, lungs and heart. Presence of autoantibodies reactive against multiple cellular proteins is a characteristic feature of SLE. Autoantibody deposition in the kidneys, often leads to their failure and is a major complication of SLE. Activation of autoreactive T cells and their interaction with B cells is a hallmark in the generation of autoantibodies. This study investigates the hypothesis that molecular mimicry between peptides originating from the human microbiome and self-antigens is involved in the activation of T cells reactive with lupus associated autoantigens.To investigate our hypothesis, we chose Ro60 protein as a model antigen and HLA-DR3 transgenic mice as an animal model. Autoantibodies to Ro60 are a major component of the autoantibody repertoire in SLE patients and HLA-DR3 is the highest genetic risk factor for SLE. Using information about three T cell epitopes on Ro60 and critical amino acids within those regions, pattern search analysis was carried out and several candidate peptides short listed. Synthetic peptides were obtained and tested for their ability to activate Ro60 reactive T cell hybridomas. Selected peptides were used to immunize HLA-DR3 transgenic mice and induction of antibody responses analyzed. We have identified hundreds of putative mimicry peptides. Several peptides originating from microbes residing in the human oral cavity, gut and skin were able to activate Ro60 reactive T cell hybridomas. We have also found that a mimicry peptide was able to induce immune response in HLA-DR3 transgenic mice against the peptide as well as La protein (which is associated with Ro60). The ability of whole protein to activate T cell hybridomas and induce anti-La and anti-Ro60 immune response in HLA-DR3 transgenic mice is being investigated.Our data suggests that T cells reactive against Ro60 can be activated by exposure to multiple peptides originating from different microbes that are present in the human microbiome. Interaction of these T cells with autoreactive B cells can induce autoantibodies. Thus, we would like to suggest that in some lupus patients, repeated exposure to different microbes over a long period of time can initiate autoimmune responses

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells

Estimating Methane Emissions from a Dairy Farm Using a Computer Program

The aim of our study was to estimate methane (CH4) emissions from a dairy cattle farm using a computer application. Emissions of CH4 in the air were forecast for a representative dairy farm raising Holstein-Friesian cows. The cowshed was equipped with a mechanical forced ventilation system with a centrally located ventilation duct. The volume of emissions from the emitter was established, taking into account meteorological conditions. For one year of operation of the emitter, the average annual emission was 1.301 kg/h. The maximum emission of CH4 was estimated at 3.51 kg/h. These data can be helpful in estimating the environmental burden of a dairy farm and in determining the role of ruminants in global warming

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006890.]

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

Letter from John C. Brewer to Emma Davis. The first part discusses mutual acquaintances, and John's wishes that Emma will not forget their memories together. The second part, dated for "Wednesday eve", discusses his and Miss Genie's relationship, what Miss Genie thinks of Emma, and Emma not feeling well