Illumination on the structure and characteristics of Entamoeba histolytica genome.

Entamoeba histolytica, likes other Organismes, is characterized by diversity and heterogeneity in its genetic content, which is one of the most paramount reasons for survival, and the increase in susceptibility to infection. Non-condensation of chromosomes during the process of cell division and the ambiguity of the chromosomal ploidy makes predicting the exact chromosomal numeral difficult. Genes distributed across 14 chromosomes as well as many extra-chromosome elements. Most Genes compose of one axon only, with Introns in 25% of Genes. This genome is characterized by the presence of Polymorphic internal repeat regions, and several gene families, one of these large families encoding Transmembrane kinas, Cysteine protease (CP), SREHP protein, and others.

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed Ankyrin Repeat Proteins (DARPins) are a class of antibody mimetics with a high and mostly unexplored potential in drug development. They are clinically validated and thus represent a true alternative to classical immunoglobulin formats. In contrast to immunoglobulins, they are built from solenoid protein domains comprising an N-terminal capping repeat, one or more internal repeats and a C-terminal capping repeat. By using in silico analysis and a rationally guided Ala-Scan, we identified position 17 of the N-terminal capping repeat to play a key role for the overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by about 8°C to 10°C when the original Asp17 was replaced by Leu, Val, Ile, Met, Ala or Thr, as shown by high-temperature unfolding experiments at equilibrium. We then transferred the Asp17Leu mutation to various backgrounds, including different N- and C-terminal capping repeats and clinically validated DARPin domains, such as the VEGF-binding ankyrin repeat domain of abicipar pegol. In all cases, the proteins remained monomeric and showed improvements in the thermostability of about 8°C to 16°C. Thus, the replacement of Asp17 seems to be generically applicable to this drug class. Molecular dynamics simulations show that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, such a beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development. This mutation is likely responsible, at least in part, for the very high melting temperature (>90°C) of this promising anti-Covid-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.

Illumination on the Structure and Characteristics of Entamoeba Histolytica Genome

Entamoeba histolytica, like other Organismes, is characterized by diversity and heterogeneity in its genetic content, which is one of the most important reasons for survival, and the increase in susceptibility to infection.Non-condensation of chromosomes during the process of cell division and the ambiguity of the chromosomal ploidy makes predicting the exact chromosomal number difficult. Genes distributed across 14 chromosomes as well as many extra-chromosome elements. Most Genes composed of one axon only, with Introns in 25% of Genes. This genome is characterized by the presence of Polymorphic internal repeat regions, and several gene families, one of these large families encoding Transmembrane kinas, Cysteine protease (CP), SREHP protein, and others.

Marek's disease virus requires both copies of the inverted repeat regions for efficient in vivo replication and pathogenesis

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of chickens. The MDV genome consists of two unique regions that are both flanked by inverted repeat regions. These repeats harbor several genes involved in virus replication and pathogenesis, but it remains unclear why MDV and other herpesviruses harbor these large sequence duplications. In this study, we set to determine if both copies of these repeat regions are required for MDV replication and pathogenesis. Our results demonstrate that MDV mutants lacking the entire internal repeat region (ΔIRLS) efficiently replicate and spread from cell-to-cell in vitro. However, ΔIRLS replication was severely impaired in infected chickens and the virus caused significantly less frequent disease and tumors compared to the controls. In addition, we also generated recombinant viruses that harbor a deletion of most of the internal repeat region, leaving only short terminal sequences behind (ΔIRLS-HR). These remaining homologous sequences facilitated rapid restoration of the deleted repeat region, resulting in a virus that caused disease and tumors comparable to the wild type. Therefore, ΔIRLS-HR represents an excellent platform for rapid genetic manipulation of the virus genome in the repeat regions. Taken together, our study demonstrates that MDV requires both copies of the repeats for efficient replication and pathogenesis in its natural host. IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes losses in the poultry industry of up to $2 billion per year. The virus is also widely used as a model to study alphaherpesvirus pathogenesis and virus-induced tumor development in a natural host. MDV and most other herpesviruses harbor direct or inverted repeats regions in their genome. However, the role of these sequence duplications in MDV remains elusive and has never been investigated in a natural virus-host model for any herpesvirus. Here, we demonstrate that both copies of the repeats are needed for efficient MDV replication and pathogenesis in vivo, while replication was not affected in cell culture. With this, we further dissect herpesvirus genome biology and the role of repeat regions in Marek's disease virus replication and pathogenesis.

UvCom1 Is an Important Regulator Required for Development and Infection in the Rice False Smut Fungus Ustilaginoidea virens

Ustilaginoidea virens is an economically important biotrophic fungal pathogen that causes rice false smut worldwide. However, the regulatory mechanisms of smut ball formation under U. virens infection remain unclear. Here, we identified an important transcription factor, UvCom1, from this pathogen, which regulates the formation of smut balls on rice spikelets. UvCom1 contains two conserved internal repeat 1 (RPT) domains and is found only in fungi, with specific conservation in species of Basidiomycetes and Ascomycetes. UvCom1 protein N- or C-terminal fusion vectors both showed transactivation activity in yeast. Deletion of UvCom1 significantly affected the vegetative growth and conidiation of U. virens. UvCom1 negatively regulated the responses to oxidative, osmotic, and cell wall stresses. Remarkably, UvCom1 was found to be essential for the formation of rice smut balls, and UvCom1 deletion mutants lost the ability to stably utilize nutrients from the rice host. UvCom1 was also highly expressed at the mycelial expansion stage. Transcriptomic analysis and quantitative real-time PCR revealed that UvCom1 could affect the expression of genes significantly enriched in transmembrane transport. This study demonstrates that UvCom1 is a key transcription factor governing smut ball formation of this biotrophic fungus.

Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance

ABSTRACTKaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA’s ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCEKSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1—and, by extension, EBV—diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC). IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

tale-Based Genetic Diversity of Chinese Isolates of the Citrus Canker Pathogen Xanthomonas citri subsp. citri

Pathotype A of Xanthomonas citri subsp. citri, the cause of citrus bacterial canker (CBC), is assumed to have originated in southern China. PthA, a type III secreted transcriptional activator-like effector (TALE), is a major pathogenicity determinant in X. citri subsp. citri. To investigate the diversity of X. citri subsp. citri in China, genomic and plasmid DNA of 105 X. citri subsp. citri isolates, collected from nine citrus-growing provinces of China, were digested by BamHI and hybridized with an internal repeat region of pthA. Strains were classified into 14 different genotypes (designated A to N) based on the number and size of pthA homologues. Genotypes B and G represented 19 and 62% of the isolate collection, respectively. Genotypes J and L lacked pthA or a pthA-hybridizing fragment and were less virulent on grapefruit (C. paradisi) and sweet orange (C. sinensis) compared with strains containing pthA or a pthA homologue. The virulence of genotypes J and L was increased when the wild-type pthA was introduced. Genotype I, which was isolated from sweet orange in Jiangxi province, caused typical canker symptoms and may contain a novel pthA-like gene. To our knowledge, this is the first description of genetic diversity in Chinese CBC strains based on tale gene analysis.