Human otic progenitor cell models of congenital hearing loss reveal potential pathophysiologic mechanisms of Zika virus and cytomegalovirus infections.

Congenital hearing loss is a common chronic condition affecting children in both developed and developing nations. Viruses correlated with congenital hearing loss include human cytomegalovirus (HCMV) and Zika virus (ZIKV), which causes congenital Zika syndrome. The mechanisms by which HCMV and ZIKV infections cause hearing loss are poorly understood. It is challenging to study human inner ear cells because they are encased in bone and also scarce as autopsy samples. Recent advances in culturing human stem cell-derived otic progenitor cells (OPCs) have allowed us herein to describe successful in vitro infection of OPCs with HCMV and ZIKV, and also to propose potential mechanisms by which each viral infection could affect hearing. We find that ZIKV infection rapidly and significantly induces the expression of type I interferon and interferon-stimulated genes, while OPC viability declines, at least in part, from apoptosis. In contrast, HCMV infection did not appear to upregulate interferons or cause a reduction in cell viability, and instead disrupted expression of key genes and pathways associated with inner ear development and function, including Cochlin, nerve growth factor receptor, SRY-box transcription factor 11, and transforming growth factor-beta signaling. These findings suggest that ZIKV and HCMV infections cause congenital hearing loss through distinct pathways, that is, by inducing progenitor cell death in the case of ZIKV infection, and by disruption of critical developmental pathways in the case of HCMV infection. IMPORTANCE: Congenital virus infections inflict substantial morbidity and devastating disease in neonates worldwide, and hearing loss is a common outcome. It has been difficult to study viral infections of the human hearing apparatus because it is embedded in the temporal bone of the skull. Recent technological advances permit the differentiation of otic progenitor cells (OPCs) from human-induced pluripotent stem cells. This paper is important for demonstrating that inner ear virus infections can be modeled in vitro using OPCs. We infected OPCs with two viruses associated with congenital hearing loss: human cytomegalovirus (HCMV), a DNA virus, or Zika virus (ZIKV), an RNA virus. An important result is that the gene expression and cytokine production profiles of HCMV/ZIKV-infected OPCs are markedly dissimilar, suggesting that mechanisms of hearing loss are also distinct. The specific molecular regulatory pathways identified in this work could suggest important targets for therapeutics.

FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation.

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.

Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral translation and Nsp1 evasion.

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction.

BACKGROUND: COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. METHODS: We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. RESULTS: We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. CONCLUSIONS: Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis. ONE SENTENCE SUMMARY: Antibody-mediated SARS-CoV-2 infection of monocytes activates inflammation and cytokine release.

The oak gene expression atlas: insights into Fagaceae genome evolution and the discovery of genes regulated during bud dormancy release.

BACKGROUND: Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. RESULTS: The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. CONCLUSION: In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination.

BACKGROUND: The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. RESULTS: In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. CONCLUSION: This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Transcriptional profiling of bud dormancy induction and release in oak by next-generation sequencing.

BACKGROUND: In temperate regions, the time lag between vegetative bud burst and bud set determines the duration of the growing season of trees (i.e. the duration of wood biomass production). Dormancy, the period during which the plant is not growing, allows trees to avoid cold injury resulting from exposure to low temperatures. An understanding of the molecular machinery controlling the shift between these two phenological states is of key importance in the context of climatic change. The objective of this study was to identify genes upregulated during endo- and ecodormancy, the two main stages of bud dormancy. Sessile oak is a widely distributed European white oak species. A forcing test on young trees was first carried out to identify the period most likely to correspond to these two stages. Total RNA was then extracted from apical buds displaying endo- and ecodormancy. This RNA was used for the generation of cDNA libraries, and in-depth transcriptome characterization was performed with 454 FLX pyrosequencing technology. RESULTS: Pyrosequencing produced a total of 495,915 reads. The data were cleaned, duplicated reads removed, and sequences were mapped onto the oak UniGene data. Digital gene expression analysis was performed, with both R statistics and the R-Bioconductor packages (edgeR and DESeq), on 6,471 contigs with read numbers ≥ 5 within any contigs. The number of sequences displaying significant differences in expression level (read abundance) between endo- and ecodormancy conditions ranged from 75 to 161, depending on the algorithm used. 13 genes displaying significant differences between conditions were selected for further analysis, and 11 of these genes, including those for glutathione-S-transferase (GST) and dehydrin xero2 (XERO2) were validated by quantitative PCR. CONCLUSIONS: The identification and functional annotation of differentially expressed genes involved in the "response to abscisic acid", "response to cold stress" and "response to oxidative stress" categories constitutes a major step towards characterization of the molecular network underlying vegetative bud dormancy, an important life history trait of long-lived organisms.

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak.

BACKGROUND: The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity. RESULTS: We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html. CONCLUSIONS: This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.

RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain-containing protein.

Restriction of long-distance movement of several potyviruses in Arabidopsis (Arabidopsis thaliana) is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2, and RTM3. RTM1 encodes a protein belonging to the jacalin family, and RTM2 encodes a protein that has similarities to small heat shock proteins. In this article, we describe the positional cloning of RTM3, which encodes a protein belonging to an undescribed protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its amino-terminal region and a coiled-coil domain at its carboxy-terminal end. Involvement in the RTM resistance system is the first biological function experimentally identified for a member of this new gene family in plants. Our analyses showed that the coiled-coil domain is not only highly conserved between RTM3-homologous MATH-containing proteins but also in proteins lacking a MATH domain. The cluster organization of the RTM3 homologs in the Arabidopsis genome suggests the role of duplication events in shaping the evolutionary history of this gene family, including the possibility of deletion or duplication of one or the other domain. Protein-protein interaction experiments revealed RTM3 self-interaction as well as an RTM1-RTM3 interaction. However, no interaction has been detected involving RTM2 or the potyviral coat protein previously shown to be the determinant necessary to overcome the RTM resistance. Taken together, these observations strongly suggest the RTM proteins might form a multiprotein complex in the resistance mechanism to block the long-distance movement of potyviruses.

"Contrasting patterns of selection at Pinus pinaster Ait. Drought stress candidate genes as revealed by genetic differentiation analyses".

The importance of natural selection for shaping adaptive trait differentiation among natural populations of allogamous tree species has long been recognized. Determining the molecular basis of local adaptation remains largely unresolved, and the respective roles of selection and demography in shaping population structure are actively debated. Using a multilocus scan that aims to detect outliers from simulated neutral expectations, we analyzed patterns of nucleotide diversity and genetic differentiation at 11 polymorphic candidate genes for drought stress tolerance in phenotypically contrasted Pinus pinaster Ait. populations across its geographical range. We compared 3 coalescent-based methods: 2 frequentist-like, including 1 approach specifically developed for biallelic single nucleotide polymorphisms (SNPs) here and 1 Bayesian. Five genes showed outlier patterns that were robust across methods at the haplotype level for 2 of them. Two genes presented higher F(ST) values than expected (PR-AGP4 and erd3), suggesting that they could have been affected by the action of diversifying selection among populations. In contrast, 3 genes presented lower F(ST) values than expected (dhn-1, dhn2, and lp3-1), which could represent signatures of homogenizing selection among populations. A smaller proportion of outliers were detected at the SNP level suggesting the potential functional significance of particular combinations of sites in drought-response candidate genes. The Bayesian method appeared robust to low sample sizes, flexible to assumptions regarding migration rates, and powerful for detecting selection at the haplotype level, but the frequentist-like method adapted to SNPs was more efficient for the identification of outlier SNPs showing low differentiation. Population-specific effects estimated in the Bayesian method also revealed populations with lower immigration rates, which could have led to favorable situations for local adaptation. Outlier patterns are discussed in relation to the different genes' putative involvement in drought tolerance responses, from published results in transcriptomics and association mapping in P. pinaster and other related species. These genes clearly constitute relevant candidates for future association studies in P. pinaster.