Dipeptidyl peptidase 4 interacts with porcine coronavirus PHEV spikes and mediates host range expansion.

Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, is prevalent in natural reservoir pigs and infects mice. This raises concerns about host jumping or spillover, but little is known about the cause of occurrence. Here, we revealed that dipeptidyl peptidase 4 (DPP4) is a candidate binding target of PHEV spikes and works as a broad barrier to overcome. Investigations of the host breadth of PHEV confirmed that cells derived from pigs and mice are permissive to virus propagation. Both porcine DPP4 and murine DPP4 have high affinity for the viral spike receptor-binding domain (RBD), independent of their catalytic activity. Loss of DPP4 expression results in limited PHEV infection. Structurally, PHEV spike protein binds to the outer surface of blades IV and V of the DPP4 ß-propeller domain, and the DPP4 residues N229 and N321 (relative to human DPP4 numbering) participate in RBD binding via its linked carbohydrate entities. Removal of these N-glycosylations profoundly enhanced the RBD-DPP4 interaction and viral invasion, suggesting they act as shielding in PHEV infection. Furthermore, we found that glycosylation, rather than structural differences or surface charges, is more responsible for DPP4 recognition and species barrier formation. Overall, our findings shed light on virus-receptor interactions and highlight that PHEV tolerance to DPP4 orthologs is a putative determinant of its cross-species transmission or host range expansion.IMPORTANCEPHEV is a neurotropic betacoronavirus that is circulating worldwide and has raised veterinary and economic concerns. In addition to being a reservoir species of pigs, PHEV can also infect wild-type mice, suggesting a "host jump" event. Understanding cross-species transmission is crucial for disease prevention and control but remains to be addressed. Herein, we show that the multifunctional receptor DPP4 plays a pivotal role in the host tropism of PHEV and identifies the conserved glycosylation sites in DPP4 responsible for this restriction. These findings highlight that the ability of PHEV to utilize DPP4 orthologs potentially affects its natural host expansion.

Benign mixed mammary tumor with sebaceous differentiation in a dog.

We describe here a case of canine mammary benign mixed tumor with sebaceous metaplasia in the right fifth mammary gland of an eight-year-old, intact female Poodle dog. Grossly, the mass was firm with off-white, poorly lobulated cut surfaces. Histologically, the luminal epithelial cells and myoepithelial cells proliferated with cartilage formation and focal squamous metaplasia. Moreover, a large number of nests of various sizes, which were filled with foamy cells in the center and associated with basaloid reserve-like cells in the periphery, showed sebaceous gland-like structures. Immunohistochemically, myoepithelial cells and reserve-like cells in the metaplastic sebaceous gland-like structures were CK14, α-smooth muscle actin (α-SMA) and p63 positive, suggesting a possibility that these two components may have a common cell of origin.

mTOR hypoactivity leads to trophectoderm cell failure by enhancing lysosomal activation and disrupting the cytoskeleton in preimplantation embryo.

BACKGROUND: Metabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood. METHODS: Pregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos. RESULTS: We showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts. CONCLUSIONS: These results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

The role of lysosomes as intermediates in betacoronavirus PHEV egress from nerve cells.

IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate ßCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.

Sex differences in paternal arsenic-induced intergenerational metabolic effects are mediated by estrogen.

BACKGROUND: Gene-environment interactions contribute to metabolic disorders such as diabetes and dyslipidemia. In addition to affecting metabolic homeostasis directly, drugs and environmental chemicals can cause persistent alterations in metabolic portfolios across generations in a sex-specific manner. Here, we use inorganic arsenic (iAs) as a prototype drug and chemical to dissect such sex differences. METHODS: After weaning, C57BL/6 WT male mice were treated with 250 ppb iAs in drinking water (iAsF0) or normal water (conF0) for 6 weeks and then bred with 15-week-old, non-exposed females for 3 days in cages with only normal water (without iAs), to generate iAsF1 or conF1 mice, respectively. F0 females and all F1 mice drank normal water without iAs all the time. RESULTS: We find that exposure of male mice to 250 ppb iAs leads to glucose intolerance and insulin resistance in F1 female offspring (iAsF1-F), with almost no change in blood lipid profiles. In contrast, F1 males (iAsF1-M) show lower liver and blood triglyceride levels than non-exposed control, with improved glucose tolerance and insulin sensitivity. The liver of F1 offspring shows sex-specific transcriptomic changes, with hepatocyte-autonomous alternations of metabolic fluxes in line with the sex-specific phenotypes. The iAsF1-F mice show altered levels of circulating estrogen and follicle-stimulating hormone. Ovariectomy or liver-specific knockout of estrogen receptor α/ß made F1 females resemble F1 males in their metabolic responses to paternal iAs exposure. CONCLUSIONS: These results demonstrate that disrupted reproductive hormone secretion in alliance with hepatic estrogen signaling accounts for the sex-specific intergenerational effects of paternal iAs exposure, which shed light on the sex disparities in long-term gene-environment interactions.

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice.

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR-/-) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR-/- mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR-/- mice, warrant further investigations.

Paternal phthalate exposure-elicited offspring metabolic disorders are associated with altered sperm small RNAs in mice.

Exposure to ubiquitous plastic-associated endocrine disrupting chemicals (EDCs) is associated with the increased risk of many chronic diseases. For example, phthalate exposure is associated with cardiometabolic mortality in humans, with societal costs ∼ $39 billion/year or more. We recently demonstrated that several widely used plastic-associated EDCs increase cardiometabolic disease in appropriate mouse models. In addition to affecting adult health, parental exposure to EDCs has also been shown to cause metabolic disorders, including obesity and diabetes, in the offspring. While most studies have focused on the impact of maternal EDC exposure on the offspring's health, little is known about the effects of paternal EDC exposure. In the current study, we investigated the adverse impact of paternal exposure to a ubiquitous but understudied phthalate, dicyclohexyl phthalate (DCHP) on the metabolic health of F1 and F2 offspring in mice. Paternal DCHP exposure led to exacerbated insulin resistance and impaired insulin signaling in F1 offspring without affecting diet-induced obesity. We previously showed that sperm small non-coding RNAs including tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs) contribute to the intergenerational transmission of paternally acquired metabolic disorders. Using a novel PANDORA-seq, we revealed that DCHP exposure can lead to sperm tsRNA/rsRNA landscape changes that were undetected by traditional RNA-seq, which may contribute to DCHP-elicited adverse effects. Lastly, we found that paternal DCHP can also cause sex-specific transgenerational adverse effects in F2 offspring and elicited glucose intolerance in female F2 descendants. Our results suggest that exposure to endocrine disrupting phthalates may have intergenerational and transgenerational adverse effects on the metabolic health of their offspring. These findings increase our understanding of the etiology of chronic human diseases originating from chemical-elicited intergenerational and transgenerational effects.

Invasive spindle-cell rhabdomyosarcoma with osteolysis in a dog: case report and literature review.

Rhabdomyosarcoma (RMS), a malignant mesenchymal neoplasm derived from skeletal muscle, is relatively rare in both human and veterinary medicine. Here we report an unusual case of invasive spindle-cell RMS (SCRMS) with bone infiltration and pathologic fracture in a 3.5-y-old intact female Bulldog. Radiographically, a large, predominantly osteolytic mass in the tibia and fibula of the left hindlimb had features typical of a malignant primary bone tumor. Clinically, osteosarcoma was suspected, and the leg was amputated. Histologically, the mass was composed of loosely interwoven spindle-cell fascicles; tumor cells were fusiform with cigar-shaped nuclei and abundant eosinophilic cytoplasm. The neoplastic cells were strongly immunopositive for vimentin, muscle-specific actin, desmin, myogenin, and myoD1. Invasive SCRMS with osteolysis was diagnosed based on the histologic examination and immunohistochemical (IHC) stains. The dog was alive without any evidence of local recurrence or distant metastasis 18 mo post-surgery. RMS should be included in the differential diagnosis when osteolysis occurs; IHC staining confirmation is of great value for definitive diagnosis and treatment planning.

Porcine Hemagglutinating Encephalomyelitis Virus Co-Opts Multivesicular-Derived Exosomes for Transmission.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the family Coronaviridae, genus Betacoronavirus, and subgenus Embecovirus that causes neurological disorders, vomiting and wasting disease (VWD), or influenza-like illness (ILI) in pigs. Exosomes regulate nearby or distant cells as a means of intercellular communication; however, whether they are involved in the transmission of viral reference materials during PHEV infection is unknown. Here, we collected exosomes derived from PHEV-infected neural cells (PHEV-exos) and validated their morphological, structural, and content characteristics. High-resolution mass spectrometry indicated that PHEV-exos carry a variety of cargoes, including host innate immunity sensors and viral ingredients. Furthermore, transwell analysis revealed that viral ingredients, such as proteins and RNA fragments, could be encapsulated in the exosomes of multivesicular bodies (MVBs) to nonpermissive microglia. Inhibition of exosome secretion could suppress PHEV infection. Therefore, we concluded that the mode of infectious transmission of PHEV is likely through a mixture of virus-modified exosomes and virions and that exosomal export acts as a host strategy to induce an innate response in replicating nonpermissive bystander cells free of immune system recognition. IMPORTANCE The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a large number of deaths worldwide. Clinical neurological complications have occurred in some cases; however, knowledge of the natural history of coronavirus in the central nervous system (CNS) is thus far limited. PHEV is a typical neurotropic betacoronavirus (ß-CoV) that propagates via neural circuits in the host CNS after peripheral incubation rather than through the bloodstream. It is therefore a good prototype pathogen to investigate the neuropathological pathogenesis of acute human coronavirus infection. In this study, we demonstrate a new association between host vesicle-based secretion and PHEV infection, showing that multivesicular-derived exosomes are one of the modes of infectious transmission and that they mediate the transfer of immunostimulatory cargo to uninfected neuroimmune cells. These findings provide novel insights into the treatment and monitoring of neurological consequences associated with ß-CoV, similar to those associated with SARS-CoV-2.

PHEV infection: A promising model of betacoronavirus-associated neurological and olfactory dysfunction.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic coronavirus belonging to the genus Betacoronavirus. Similar to pathogenic coronaviruses to which humans are susceptible, such as SARS-CoV-2, PHEV is transmitted primarily through respiratory droplets and close contact, entering the central nervous system (CNS) from the peripheral nerves at the site of initial infection. However, the neuroinvasion route of PHEV are poorly understood. Here, we found that BALB/c mice are susceptible to intranasal PHEV infection and showed distinct neurological manifestations. The behavioral study and histopathological examination revealed that PHEV attacks neurons in the CNS and causes significant smell and taste dysfunction in mice. By tracking neuroinvasion, we identified that PHEV invades the CNS via the olfactory nerve and trigeminal nerve located in the nasal cavity, and olfactory sensory neurons (OSNs) were susceptible to viral infection. Immunofluorescence staining and ultrastructural observations revealed that viral materials traveling along axons, suggesting axonal transport may engage in rapid viral transmission in the CNS. Moreover, viral replication in the olfactory system and CNS is associated with inflammatory and immune responses, tissue disorganization and dysfunction. Overall, we proposed that PHEV may serve as a potential prototype for elucidating the pathogenesis of coronavirus-associated neurological complications and olfactory and taste disorders.

Exploring the expanding universe of small RNAs.

The world of small noncoding RNAs (sncRNAs) is ever-expanding, from small interfering RNA, microRNA and Piwi-interacting RNA to the recently emerging non-canonical sncRNAs derived from longer structured RNAs (for example, transfer, ribosomal, Y, small nucleolar, small nuclear and vault RNAs), showing distinct biogenesis and functional principles. Here we discuss recent tools for sncRNA identification, caveats in sncRNA expression analysis and emerging methods for direct sequencing of sncRNAs and systematic mapping of RNA modifications that are integral to their function.

Cell-surface glycans act as attachment factors for porcine hemagglutinating encephalomyelitis virus.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and highly pathogenic in veterinary clinic. Spike (S) protein of PHEV interplays with host components to cross the plasma membrane of target cells, but characterization of its functional receptors is limited. Here, we discovered that cell-surface glycans, i.e., sialic acid (SA) and heparan sulfate (HS), act as critical interacting factors of PHEV, involving in viral attachment. As shown in glycans depletion assay, removing SA or HS from N2a cells inhibits PHEV infection. Soluble sugar monomers were utilized for competitive binding tests, and we found that both SA and HS could specifically bind to PHEV and affect the viral infectivity. Furthermore, the expression of heparan sulfate proteoglycans (HSPGs), including syndecans and glypicans, and endoglycosidase heparinase which cleaves HS were regulated by PHEV RNA replication. Together, we newly identified specificity recognition of cellular glycans and PHEV during infection, providing novel cellular targets for antiviral therapies and better understanding of pathogenesis.

The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation.

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

Origins and evolving functionalities of tRNA-derived small RNAs.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the most ancient small RNAs in all domains of life and are generated by the cleavage of tRNAs. Emerging studies have begun to reveal the versatile roles of tsRNAs in fundamental biological processes, including gene silencing, ribosome biogenesis, retrotransposition, and epigenetic inheritance, which are rooted in tsRNA sequence conservation, RNA modifications, and protein-binding abilities. We summarize the mechanisms of tsRNA biogenesis and the impact of RNA modifications, and propose how thinking of tsRNA functionality from an evolutionary perspective urges the expansion of tsRNA research into a wider spectrum, including cross-tissue/cross-species regulation and harnessing of the 'tsRNA code' for precision medicine.

PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.

Author Correction: PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.

A Correction to this paper has been published: https://doi.org/10.1038/s41556-021-00687-w.