MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

LYSET/TMEM251- a novel key component of the mannose 6-phosphate pathway.

Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose 6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).Abbreviations: GlcNAc-1-PT: GlcNAc-1-phosphotransferase; KO: knockout; LSD: lysosomal storage disorder; LYSET: lysosomal enzyme trafficking factor; M6P: mannose 6-phosphate; MPRs: mannose-6-phosphate receptors, cation-dependent or -independent; MBTPS1/site-1 protease: membrane bound transcription factor peptidase, site 1; MLII: mucolipidosis type II; WT: wild-type.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

A pan-genome data structure induced by pooled sequencing facilitates variant mining in heterogeneous germplasm.

Valuable genetic variation lies unused in gene banks due to the difficulty of exploiting heterogeneous germplasm accessions. Advances in molecular breeding, including transgenics and genome editing, present the opportunity to exploit hidden sequence variation directly. Here we describe the pan-genome data structure induced by whole-genome sequencing of pooled individuals from wild populations of Patellifolia spp., a source of disease resistance genes for the related crop species sugar beet (Beta vulgaris). We represent the pan-genome as a map of reads from pooled sequencing of a heterogeneous population sample to a reference genome, plus a BLAST data base of the mapped reads. We show that this basic data structure can be queried by reference genome position or homology to identify sequence variants present in the wild relative, at genes of agronomic interest in the crop, a process known as allele or variant mining. Further we demonstrate the possibility of cataloging variants in all Patellifolia genomic regions that have corresponding single copy orthologous regions in sugar beet. The data structure, termed a "pooled read archive," can be produced, altered, and queried using standard tools to facilitate discovery of agronomically-important sequence variation. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01308-6.

Development of an SNP Assay for Marker-Assisted Selection of Soil-Borne Rhizoctonia solani AG-2-2-IIIB Resistance in Sugar Beet.

Rhizoctonia solani, causing Rhizoctonia crown and root rot, is a major risk to sugar beet (Beta vulgaris L.) cultivation. The development of resistant varieties accelerated by marker-assisted selection is a priority of breeding programs. We report the identification of a single-nucleotide polymorphism (SNP) marker linked to Rhizoctonia resistance using restriction site-associated DNA (RAD) sequencing of two geographically discrete sets of plant materials with different degrees of resistance/susceptibility to enable a wider selection of superior genotypes. The variant calling pipeline utilized SAMtools for variant calling and the resulting raw SNPs from RAD sequencing (15,988 and 22,439 SNPs) were able to explain 13.40% and 25.45% of the phenotypic variation in the two sets of material from different sources of origin, respectively. An association analysis was carried out independently on both the datasets and mutually occurring significant SNPs were filtered depending on their contribution to the phenotype using principal component analysis (PCA) biplots. To provide a ready-to-use marker for the breeding community, a systematic molecular validation of significant SNPs distributed across the genome was undertaken to combine high-resolution melting, Sanger sequencing, and rhAmp SNP genotyping. We report that RsBv1 located on Chromosome 6 (9,000,093 bp) is significantly associated with Rhizoctonia resistance (p < 0.01) and able to explain 10% of the phenotypic disease variance. The related SNP assay is thus ready for marker-assisted selection in sugar beet breeding for Rhizoctonia resistance.

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

A memory of eS25 loss drives resistance phenotypes.

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour.

A complete and accurate genome sequence provides a fundamental tool for functional genomics and DNA-informed breeding. Here, we assemble a high-quality genome (contig N50 of 6.99 Mb) of the apple anther-derived homozygous line HFTH1, including 22 telomere sequences, using a combination of PacBio single-molecule real-time (SMRT) sequencing, chromosome conformation capture (Hi-C) sequencing, and optical mapping. In comparison to the Golden Delicious reference genome, we identify 18,047 deletions, 12,101 insertions and 14 large inversions. We reveal that these extensive genomic variations are largely attributable to activity of transposable elements. Interestingly, we find that a long terminal repeat (LTR) retrotransposon insertion upstream of MdMYB1, a core transcriptional activator of anthocyanin biosynthesis, is associated with red-skinned phenotype. This finding provides insights into the molecular mechanisms underlying red fruit coloration, and highlights the utility of this high-quality genome assembly in deciphering agriculturally important trait in apple.

Genetic and mechanistic basis for APOBEC3H alternative splicing, retrovirus restriction, and counteraction by HIV-1 protease.

Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I-VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a  counter-defense mechanism against A3H in >80% of sub-Saharan African populations.

Diversity from genes to ecosystems: A unifying framework to study variation across biological metrics and scales.

Biological diversity is a key concept in the life sciences and plays a fundamental role in many ecological and evolutionary processes. Although biodiversity is inherently a hierarchical concept covering different levels of organization (genes, population, species, ecological communities and ecosystems), a diversity index that behaves consistently across these different levels has so far been lacking, hindering the development of truly integrative biodiversity studies. To fill this important knowledge gap, we present a unifying framework for the measurement of biodiversity across hierarchical levels of organization. Our weighted, information-based decomposition framework is based on a Hill number of order q = 1, which weights all elements in proportion to their frequency and leads to diversity measures based on Shannon's entropy. We investigated the numerical behaviour of our approach with simulations and showed that it can accurately describe complex spatial hierarchical structures. To demonstrate the intuitive and straightforward interpretation of our diversity measures in terms of effective number of components (alleles, species, etc.), we applied the framework to a real data set on coral reef biodiversity. We expect our framework will have multiple applications covering the fields of conservation biology, community genetics and eco-evolutionary dynamics.

Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

Seeds exist in the vulnerable state of being unable to repair the chemical degradation all organisms suffer, which slowly ages seeds and eventually results in death. Proposed seed aging mechanisms involve all classes of biological molecules, and degradation of total RNA has been detected contemporaneously with viability loss in dry-stored seeds. To identify changes specific to mRNA, we examined the soybean (Glycine max) seed transcriptome, using new, whole-molecule sequencing technology. We detected strong evidence of transcript fragmentation in 23-year-old, compared with 2-year-old, seeds. Transcripts were broken non-specifically, and greater fragmentation occurred in longer transcripts, consistent with the proposed mechanism of molecular fission by free radical attack at random bases. Seeds died despite high integrity of short transcripts, indicating that functions encoded by short transcripts are not sufficient to maintain viability. This study provides an approach to probe the asymptomatic phase of seed aging, namely by quantifying transcript degradation as a function of storage time.

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism.

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity.

APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization.

The APOBEC3 DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several APOBEC3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) shows strong virus restriction activities in a variety of experimental systems, and is subjected to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity and active nuclear import. Here we ask whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells.

Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential.

This study investigates the relationship between germination ability and damage to RNA in soybean seeds (cv 'Williams 82') stored dry at 5 °C for 1-27 years. Total germination of 14 age cohorts harvested between 2015 and 1989 ranged from 100% to 3%. Germination decline followed classic seed viability kinetics, with symptomatic seed aging beginning after 17 years of storage. RNA integrity was assessed in dry seeds by electrophoresis of total RNA, followed by calculation of the RNA integrity number (RIN, Agilent Bioanalyzer software), which evaluates RNA fragment size distributions. Analysis of RNA extracted from cotyledons, embryonic axes, plumules, and seed coats across the range of age cohorts showed consistent RNA degradation: older seeds had over-representation of small RNAs compared with younger seeds, which had nearly a 2:1 ratio of 25S and 18S rRNAs. RIN values for cotyledons and embryonic axes from the same seed were correlated. Decline in RIN tracked reduced germination, with a pronounced decrease in RIN after 17 years of storage. This led to a high correlation between the mean RIN of cotyledon RNA and the total germination percentage (R2=0.91, P<0.0001). Despite this relationship, germinable and non-germinable seeds within cohorts could not be distinguished unless the RIN was <3.5, indicating substantial deterioration. Our work demonstrates that seed RNA incurs damage over time, observable in fragment size distributions. Under the experimental conditions used here, RIN appears to be a promising surrogate for germination tests used to monitor viability of stored seeds.

Phylogeography of the wild and cultivated stimulant plant qat (Catha edulis, Celastraceae) in areas of historical cultivation.

PREMISE OF THE STUDY: Qat (Catha edulis, Celastraceae) is a woody plant species cultivated for its stimulant alkaloids. Qat is important to the economy and culture in large regions of Ethiopia, Kenya, and Yemen. Despite the importance of this species, the wild origins and dispersal of cultivars have only been described in often contradictory historical documents. We examined the wild origins, human-mediated dispersal, and genetic divergence of cultivated qat compared to wild qat. METHODS: We sampled 17 SSR markers and 1561 wild and cultivated individuals across the historical areas of qat cultivation. KEY RESULTS: On the basis of genetic structure inferred using Bayesian and nonparametric methods, two centers of origin in Kenya and one in Ethiopia were found for cultivated qat. The centers of origin in Ethiopia and northeast of Mt. Kenya are the primary sources of cultivated qat genotypes. Qat cultivated in Yemen is derived from Ethiopian genotypes rather than Yemeni wild populations. Cultivated qat with a wild Kenyan origin has not spread to Ethiopia or Yemen, whereas a small minority of qat cultivated in Kenya originated in Ethiopia. Hybrid genotypes with both Ethiopian and Kenyan parentage are present in northern Kenya. CONCLUSIONS: Ethiopian cultivars have diverged from their wild relatives, whereas Kenyan qat has diverged less. This pattern of divergence could be caused by the extinction of the wild-source qat populations in Ethiopia due to deforestation, undersampling, and/or artificial selection for agronomically important traits.

Reassessing APOBEC3G Inhibition by HIV-1 Vif-Derived Peptides.

The human APOBEC3G (A3G) enzyme restricts HIV-1 in the absence of the viral accessory protein viral infectivity factor (Vif) by deaminating viral cDNA cytosines to uracils. These uracil lesions base-pair with adenines during the completion of reverse transcription and result in A3G signature G-to-A mutations in the viral genome. Vif protects HIV-1 from A3G-mediated restriction by forming an E3-ubiquitin ligase complex to polyubiquitinate A3G and trigger its degradation. Prior studies indicated that Vif may also directly block the enzymatic activity of A3G and, provocatively, that Vif-derived peptides, Vif 25-39 and Vif 105-119, are similarly inhibitory. Here, we show that Vif 25-39 does not inhibit A3G enzymatic activity and that the inhibitory effect of Vif 105-119 and that of a shorter derivative Vif 107-115, although recapitulated, are non-specific. We also elaborate a simple method for assaying DNA cytosine deaminase activity that eliminates potential polymerase chain reaction-induced biases. Our results show that these Vif-derived peptides are unlikely to be useful as tools to study A3G function or as leads for the development of future therapeutics.

Geography of Genetic Structure in Barley Wild Relative Hordeum vulgare subsp. spontaneum in Jordan.

Informed collecting, conservation, monitoring and utilization of genetic diversity requires knowledge of the distribution and structure of the variation occurring in a species. Hordeum vulgare subsp. spontaneum (K. Koch) Thell., a primary wild relative of barley, is an important source of genetic diversity for barley improvement and co-occurs with the domesticate within the center of origin. We studied the current distribution of genetic diversity and population structure in H. vulgare subsp. spontaneum in Jordan and investigated whether it is correlated with either spatial or climatic variation inferred from publically available climate layers commonly used in conservation and ecogeographical studies. The genetic structure of 32 populations collected in 2012 was analyzed with 37 SSRs. Three distinct genetic clusters were identified. Populations were characterized by admixture and high allelic richness, and genetic diversity was concentrated in the northern part of the study area. Genetic structure, spatial location and climate were not correlated. This may point out a limitation in using large scale climatic data layers to predict genetic diversity, especially as it is applied to regional genetic resources collections in H. vulgare subsp. spontaneum.

Chloroplast heterogeneity and historical admixture within the genus Malus.

UNLABELLED: • PREMISE OF THE STUDY: The genus Malus represents a unique and complex evolutionary context in which to study domestication. Several Malus species have provided novel alleles and traits to the cultivars. The extent of admixture among wild Malus species has not been well described, due in part to limited sampling of individuals within a taxon.• METHODS: Four chloroplast regions (1681 bp total) were sequenced and aligned for 412 Malus individuals from 30 species. Phylogenetic relationships were reconstructed using maximum parsimony. The distribution of chloroplast haplotypes among species was examined using statistical parsimony, phylogenetic trees, and a median-joining network.• KEY RESULTS: Chloroplast haplotypes are shared among species within Malus. Three major haplotype-sharing networks were identified. One includes species native to China, Western North America, as well as Malus domestica Borkh, and its four primary progenitor species: M. sieversii (Ledeb.) M. Roem., M. orientalis Uglitzk., M. sylvestris (L.) Mill., and M. prunifolia (Willd.) Borkh; another includes five Chinese Malus species, and a third includes the three Malus species native to Eastern North America.• CONCLUSIONS: Chloroplast haplotypes found in M. domestica belong to a single, highly admixed network. Haplotypes shared between the domesticated apple and its progenitors may reflect historical introgression or the retention of ancestral polymorphisms. Multiple individuals should be sampled within Malus species to reveal haplotype heterogeneity, if complex maternal contributions to named species are to be recognized.

Genetic diversity in Malus ×domestica (Rosaceae) through time in response to domestication.

UNLABELLED: • PREMISE OF THE STUDY: Patterns of genetic diversity in domesticated plants are affected by geographic region of origin and cultivation, intentional artificial selection, and unintentional genetic bottlenecks. While bottlenecks are mainly associated with the initial domestication process, they can also affect diversity during crop improvement. Here, we investigate the impact of the improvement process on the genetic diversity of domesticated apple in comparison with other perennial and annual fruit crops.• METHODS: Apple cultivars that were developed at various times (ranging from the 13th through the 20th century) and 11 of the 15 apple cultivars that are used for 90% of the apple production in the United States were surveyed for genetic diversity based on either 9 or 19 simple sequence repeats (SSRs). Diversity was compared using standard metrics and model-based approaches based on expected heterozygosity (He) at equilibrium. Improvement bottleneck data for fruit crops were also collected from the literature.• KEY RESULTS: Domesticated apples showed no significant reduction in genetic diversity through time across the last eight centuries. Diversity was generally high, with an average He > 0.7 for apples from all centuries. However, diversity of the apples currently used for the bulk of commercial production was lower.• CONCLUSIONS: The improvement bottleneck in domesticated apples appears to be mild or nonexistent, in contrast to improvement bottlenecks in many annual and perennial fruit crops, as documented from the literature survey. The low diversity of the subset of cultivars used for commercial production, however, indicates that an improvement bottleneck may be in progress for this perennial crop.