Terminal Repeat Research Articles

Polyploids of Brassicaceae: Genomic Insights and Assembly Strategies.

The Brassicaceae family is distinguished by its inclusion of high-value crops such as cabbage, broccoli, mustard, and wasabi, all noted for their glucosinolates. In this family, many polyploidy species are distributed and shaped by numerous whole-genome duplications, independent genome doublings, and hybridization events. The evolutionary trajectory of the family is marked by enhanced diversification and lineage splitting after paleo- and meso-polyploidization, with discernible remnants of whole-genome duplications within their genomes. The recent neopolyploidization events notably increased the proportion of polyploid species within the family. Although sequencing efforts for the Brassicaceae genome have been robust, accurately distinguishing sub-genomes remains a significant challenge, frequently complicating the assembly process. Assembly strategies include comparative analyses with ancestral species and examining k-mers, long terminal repeat retrotransposons, and pollen sequencing. This review comprehensively explores the unique genomic characteristics of the Brassicaceae family, with a particular emphasis on polyploidization events and the latest strategies for sequencing and assembly. This review will significantly improve our understanding of polyploidy in the Brassicaceae family and assist in future genome assembly methods.

Actively Expressed Intergenic Genes Generated by Transposable Element Insertions in Gossypium hirsutum Cotton.

The genomes and annotated genes of allotetraploid cotton Gossypium hirsutum have been extensively studied in recent years. However, the expression, regulation, and evolution of intergenic genes (ITGs) have not been completely deciphered. In this study, we identified a novel set of actively expressed ITGs in G. hirsutum cotton, through transcriptome profiling based on deep sequencing data, as well as chromatin immunoprecipitation, followed by sequencing (ChIP-seq) of histone modifications and how the ITGs evolved. Totals of 17,567 and 8249 ITGs were identified in G. hirsutum and Gossypium arboreum, respectively. The expression of ITGs in G. hirsutum was significantly higher than that in G. arboreum. Moreover, longer exons were observed in G. hirsutum ITGs. Notably, 42.3% of the ITGs from G. hirsutum were generated by the long terminal repeat (LTR) insertions, while their proportion in genic genes was 19.9%. The H3K27ac and H3K4me3 modification proportions and intensities of ITGs were equivalent to genic genes. The H3K4me1 modifications were lower in ITGs. Additionally, evolution analyses revealed that the ITGs from G. hirsutum were mainly produced around 6.6 and 1.6 million years ago (Mya), later than the pegged time for genic genes, which is 7.0 Mya. The characterization of ITGs helps to elucidate the evolution of cotton genomes and shed more light on their biological functions in the transcriptional regulation of eukaryotic genes, along with the roles of histone modifications in speciation and diversification.

Chromosome-scale genome assembly and annotation of the tetraploid potato cultivar Diacol Capiro adapted to the Andean region.

Potato (Solanum tuberosum) is an essential crop for food security and is ranked as the third most important crop worldwide for human consumption. The Diacol Capiro cultivar holds the dominant position in Colombian cultivation, primarily catering to the food processing industry. This highly heterozygous, autotetraploid cultivar belongs to the Andigenum group and it stands out for its adaptation to a wide variety of environments spanning altitudes from 1,800 to 3,200 meters above sea level. Here, a chromosome-scale assembly, referred to as DC, is presented for this cultivar. The assembly was generated by combining circular consensus sequencing with proximity ligation Hi-C for the scaffolding and represents 2.369 Gb with 48 pseudochromosomes covering 2,091 Gb and an anchor rate of 88.26%. The reference genome metrics, including an N50 of 50.5 Mb, a BUSCO (Benchmarking Universal Single-Copy Orthologue) score of 99.38%, and an Long Terminal Repeat Assembly Index score of 13.53, collectively signal the achieved high assembly quality. A comprehensive annotation yielded a total of 154,114 genes, and the associated BUSCO score of 95.78% for the annotated sequences attests to their completeness. The number of predicted NLR (Nucleotide-Binding and Leucine-Rich-Repeat genes) was 2107 with a large representation of NBARC (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4) containing domains (99.85%). Further comparative analysis of the proposed annotation-based assembly with high-quality known potato genomes, showed a similar genome metrics with differences in total gene numbers related to the ploidy status. The genome assembly and annotation of DC presented in this study represent a valuable asset for comprehending potato genetics. This resource aids in targeted breeding initiatives and contributes to the creation of enhanced, resilient, and more productive potato varieties, particularly beneficial for countries in Latin America.

The genome of Vicia sativa ssp. amphicarpa provides insights into the role of terpenoids in antimicrobial resistance within subterranean fruits.

Vicia sativa ssp. amphicarpa is a unique forage crop capable of simultaneously producing fruits above and below ground, representing a typical amphicarpic plant. In this study, we sequenced and assembled seven pseudo-chromosomes of the genome of V. sativa ssp. amphicarpa (n = 7) yielding a genome size of 1.59 Gb, with a total annotation of 48 932 protein-coding genes. Long terminal repeat (LTR) elements constituted 62.28% of the genome, significantly contributing to the expansion of genome size. Phylogenetic analysis revealed that the divergence between V. sativa ssp. amphicarpa and V. sativa was around 0.88 million years ago (MYA). Comparative transcriptomic and metabolomic analysis of aerial and subterranean pod shells showed biosynthesis of terpenoids in the subterranean pod shells indicating a correlation between the antimicrobial activity of subterranean pod shells and the biosynthesis of terpenoids. Furthermore, functional validation indicates that overexpression of VsTPS5 and VsTPS16 enhances terpenoid biosynthesis for antibacterial activity. Metabolomic analysis suggests the involvement of terpenoids in the antimicrobial properties of subterranean pod shells. Deciphering the genome of V. sativa ssp. amphicarpa elucidated the molecular mechanisms behind the antimicrobial properties of subterranean fruits in amphicarpic plants, providing valuable insights for the study of amphicarpic plant biology.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Complete genome sequence of Microbacterium foliorum phage Curie, a podovirus isolated from soil in Spokane, Washington.

Bacteriophage Curie is a podovirus that infects Microbacterium foliorum. The Curie genome spans 16,810 bp, has 90 bp terminal inverted repeats, and includes 23 protein-coding genes. Its genome architecture resembles phage PineapplePizza and other phi29-like phages. Together, Curie and PineapplePizza form a new actinobacteriophage Cluster GI.

Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A

AbstractFollowing systemically administered adeno-associated virus gene therapy, vector particles are widely distributed, raising concerns about horizontal or germline vector transmission. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n = 134) received a single 6 × 1013 vector genome (vg)/kg infusion and were assessed over 3 years. Vector DNA was measured using 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative polymerase chain reaction (qPCR). Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vgs and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multicolor digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by ≤12 weeks in plasma and semen. Predominant vector genome forms transitioned from noncontiguous to full-length over time in whole blood and PBMCs, indicating formation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with steady clearance of total and encapsidated vector DNA from shedding matrices, indicates transmission risk is low. This trial was registered at www.ClinicalTrials.gov as #NCT03370913.

The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.

Highly contiguous genome assembly of Drosophila prolongata-a model for evolution of sexual dimorphism and male-specific innovations.

Drosophila prolongata is a member of the melanogaster species group and rhopaloa subgroup native to the subtropical highlands of Southeast Asia. This species exhibits an array of recently evolved male-specific morphological, physiological, and behavioral traits that distinguish it from its closest relatives, making it an attractive model for studying the evolution of sexual dimorphism and testing theories of sexual selection. The lack of genomic resources has impeded the dissection of the molecular basis of sex-specific development and behavior in this species. To address this, we assembled the genome of D. prolongata using long-read sequencing and Hi-C scaffolding, resulting in a highly complete and contiguous (scaffold N50 2.2 Mb) genome assembly of 220 Mb. The repetitive content of the genome is 24.6%, the plurality of which are long terminal repeats retrotransposons (33.2%). Annotations based on RNA-seq data and homology to related species revealed a total of 19,330 genes, of which 16,170 are protein-coding. The assembly includes 98.5% of Diptera BUSCO genes, including 93.8% present as a single copy. Despite some likely regional duplications, the completeness of this genome suggests that it can be readily used for gene expression, genome-wide association studies (GWAS), and other genomic analyses.

Genome Assembly and Structural Variation Analysis of Luffa acutangula Provide Insights on Flowering Time and Ridge Development.

Luffa spp. is an important worldwide cultivated vegetable and medicinal plant from the Cucurbitaceae family. In this study, we report a high-quality chromosome-level genome of the high-generation inbred line SG261 of Luffa acutangula. The genomic sequence was determined by PacBio long reads, Hi-C sequencing reads, and 10× Genomics sequencing, with an assembly size of 739.82 Mb, contig N50 of 18.38 Mb, and scaffold N50 of 56.08 Mb. The genome of L. acutangula SG261 was predicted to contain 27,312 protein-coding genes and 72.56% repetitive sequences, of which long terminal repeats (LTRs) were an important form of repetitive sequences, accounting for 67.84% of the genome. Phylogenetic analysis reveals that L. acutangula evolved later than Luffa cylindrica, and Luffa is closely related to Momodica charantia. Comparing the genome of L. acutangula SG261 and L. cylindrica with PacBio data, 67,128 high-quality structural variations (SVs) and 55,978 presence-absence variations (PAVs) were identified in SG261, resulting in 2424 and 1094 genes with variation in the CDS region, respectively, and there are 287 identical genes affected by two different structural variation analyses. In addition, we found that the transcription factor FY (FLOWERING LOCUS Y) families had a large expansion in L. acutangula SG261 (flowering in the morning) compared to L. cylindrica (flowering in the afternoon), which may result in the early flowering time in L. acutangula SG261. This study provides valuable reference for the breeding of and pan-genome research into Luffa species.

An optimal promoter regulating cytokine transgene expression is crucial for safe and effective oncolytic virus immunotherapy

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive “conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)” (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active “cytomegalovirus enhancer and β-actin promoter”, provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active “Rous sarcoma virus long terminal repeat”, not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

High-quality reference genome decoding and population evolution analysis of prickly Sechium edule

Sechium edule (chayote) is an important vegetable crop belonging to the Cucurbitaceae family. To decipher the chayote genome, a high-quality chromosome-level chayote genome was obtained by genome sequencing and bioinformatic analysis. The total length was 612.91 Mb, and 25 755 genes were detected in the chayote genome. The contig N50 was more than 20.01 Mb, and the scaffold N50 was over 47.11 Mb. Of the genome, 60.35% were composed of repetitive sequences, and 31.18% of genome sequences belonged to long-terminal repeats. A global alignment of homologous regions in chayote and other Cucurbitaceae plant genomes was constructed using grape as a reference. Based on this genome-wide and global alignment map, researchers can easily identify homologous collinear genes of the studied genomes in most Cucurbitaceae species. Twenty-five chayote accessions were divided into two subgroups based on phylogenetic tree, population structure analysis, and principal component analysis using genome re-sequencing data. The chayote genome, re-sequencing dataset, and comprehensive genomic analysis will accelerate comparative and functional genomic analysis of chayote and other Cucurbitaceae species in the future.

Chromosome-Scale Genome Assembly for Soft-Stem Bulrush (Schoenoplectus tabernaemontani) Confirms a Clade-Specific Whole-Genome Duplication in Cyperaceae.

Schoenoplectus tabernaemontani (C. C. Gmelin) Palla is a typical macrophyte in diverse wetland ecosystems. This species holds great potential in decontamination applications and carbon sequestration. Previous studies have shown that this species may have experienced recent polyploidization. This would make S. tabernaemontani a unique model to study the processes and consequences of whole-genome duplications in the context of the well-documented holocentric chromosomes and dysploidy events in Cyperaceae. However, the inference was not completely solid because it lacked homology information that is essential to ascertain polyploidy. We present here the first chromosome-level genome assembly for S. tabernaemontani. By combining Oxford Nanopore Technologies (ONT) long reads and Illumina short reads, plus chromatin conformation via the Hi-C method, we assembled a genome spanning 507.96 Mb, with 99.43% of Hi-C data accurately mapped to the assembly. The assembly contig N50 value was 3.62 Mb. The overall BUSCO score was 94.40%. About 68.94% of the genome was comprised of repetitive elements. A total of 36,994 protein-coding genes were predicted and annotated. Long terminal repeat retrotransposons accounted for ∼26.99% of the genome, surpassing the content observed in most sequenced Cyperid genomes. Our well-supported haploid assembly comprised 21 pseudochromosomes, each harboring putative holocentric centromeres. Our findings corroborated a karyotype of 2n = 2X = 42. We also confirmed a recent whole-genome duplication occurring after the divergence between Schoenoplecteae and Bolboschoeneae. Our genome assembly expands the scope of sequenced genomes within the Cyperaceae family, encompassing the fifth genus. It also provides research resources on Cyperid evolution and wetland conservation.

LTR retrotransposon-derived LncRNA LINC01446 promotes hepatocellular carcinoma progression and angiogenesis by regulating the SRPK2/SRSF1/VEGF axis

The causal link between long terminal repeat (LTR) retrotransposon-derived lncRNAs and hepatocellular carcinoma (HCC) remains elusive and whether these cancer-exclusive lncRNAs contribute to the effectiveness of current HCC therapies is yet to explore. Here, we investigated the activation of LTR retrotransposon-derived lncRNAs in a broad range of liver diseases. We found that LTR retrotransposon-derived lncRNAs are mainly activated in HCC and is correlated with the proliferation status of HCC. Furthermore, we discovered that an LTR retrotransposon-derived lncRNA, LINC01446, exhibits specific expression in HCC. HCC patients with higher LINC01446 expression had shorter overall survival times. In vitro and in vivo assays showed that LINC01446 promoted HCC growth and angiogenesis. Mechanistically, LINC01446 bound to serine/arginine protein kinase 2 (SRPK2) and activated its downstream target, serine/arginine splicing factor 1 (SRSF1). Furthermore, activation of the SRPK2-SRSF1 axis increased the splicing and expression of VEGF isoform A165 (VEGFA165). Notably, inhibiting LINC01446 expression dramatically impaired tumor growth in vivo and resulted in better therapeutic outcomes when combined with antiangiogenic agents. In addition, we found that the transcription factor MESI2 bound to the cryptic MLT2B3 LTR promoter and drove LINC01446 transcription in HCC cells. Taken together, our findings demonstrate that LTR retrotransposon-derived LINC01446 promotes the progression of HCC by activating the SRPK2/SRSF1/VEGFA165 axis and highlight targeting LINC01446 as a potential therapeutic strategy for HCC patients.

A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations

Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences; (2) increases correctly packaged AAV payloads; and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements.

Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52-145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution.

HIV latency potential may be influenced by intra-subtype genetic differences in the viral long-terminal repeat

IntroductionElucidation of mechanisms that drive HIV latency is essential to identifying cure strategies. While host mechanisms associated with viral persistence on antiretroviral therapy (ART) have been well studied, less is known about the viral properties that influence latency. The viral promoter element, the 5’ long terminal repeat (LTR), has been shown to affect the number of latently infected cells shortly after infection. Here we investigated the role of subtype C LTR genotypic variation on the establishment of latency in a dual reporter HIV-1 infection model.MethodsThe LTR U3 and R regions from 11 women with acute/early subtype C HIV infection were cloned into the dual reporter pRGH plasmid. Latency potential was calculated based on the expression of fluorescent reporter genes in Jurkat E6–1 cells measured by flow cytometry as the proportion of latent (mCherry +ve cells)/proportion of active (eGFP +ve mCherry +ve cells) infection. Reversal of latency was performed using PMA/Ionomycin stimulation 24 hours before fixing of cells. LTR transcriptional capacity, in the presence and absence of a heterologous subtype C Tat, was measured for the same LTRs cloned into a pGL4.10 luciferase expression vector following transfection of HEK293T cells.ResultsThe majority of proviruses were latent at day 8 post-infection, yet the proportion of latently infected cells varied significantly across participants. We observed a median latent:active infection ratio of 1.79 (range 0.86–2.83) across LTRs with the hierarchy of latency potential remaining consistent across repeat experiments. The median latent:active infection ratio decreased by a median of 3-fold following PMA/Ionomycin stimulation to 0.55 (range 0.46–0.78) indicating that a proportion of latently infected cells could produce viral proteins upon activation. Latency potential did not correlate with LTR transcriptional capacity.ConclusionsWe found intra-subtype level differences in the latency potential of LTRs from South African women independent of their transcriptional capacity, suggesting that HIV-1 LTRs have intrinsic properties that influence the proportion of latently infected cells shortly after infection. The inability to reactivate viral expression in all latently infected cells supports the complex nature of mechanisms driving latency and the need for continued advancements in methods used to study these mechanisms.

Obox4 promotes zygotic genome activation upon loss of Dux.

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux-knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here, we present multiple lines of evidence that the multicopy homeobox gene, Obox4, encodes a transcription factor that is highly expressed in mouse two-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4/Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to preimplantation development.

A treasure trove of 1034 actinomycete genomes.

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.