Spatial mapping of the total transcriptome by in situ polyadenylation.

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.

Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis.

A significant fraction of sudden death in children and young adults is due to viral myocarditis, an inflammatory disease of the heart. In this study, by using integrated single-cell and spatial transcriptomics, we created a high-resolution, spatially resolved transcriptome map of reovirus-induced myocarditis in neonatal mouse hearts. We assayed hearts collected at three timepoints after infection and studied the temporal, spatial and cellular heterogeneity of host-virus interactions. We further assayed the intestine, the primary site of reovirus infection, to establish a full chronology of molecular events that ultimately lead to myocarditis. We found that inflamed endothelial cells recruit cytotoxic T cells and undergo pyroptosis in the myocarditic tissue. Analyses of spatially restricted gene expression in myocarditic regions and the border zone identified immune-mediated cell-type-specific injury and stress responses. Overall, we observed a complex network of cellular phenotypes and spatially restricted cell-cell interactions associated with reovirus-induced myocarditis in neonatal mice.

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury.

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

Mammalian orthoreovirus Infection is Enhanced in Cells Pre-Treated with Sodium Arsenite.

Following reovirus infection, cells activate stress responses that repress canonical translation as a mechanism to limit progeny virion production. Work by others suggests that these stress responses, which are part of the integrated stress response (ISR), may benefit rather than repress reovirus replication. Here, we report that compared to untreated cells, treating cells with sodium arsenite (SA) to activate the ISR prior to infection enhanced viral protein expression, percent infectivity, and viral titer. SA-mediated enhancement was not strain-specific, but was cell-type specific. While SA pre-treatment of cells offered the greatest enhancement, treatment within the first 4 h of infection increased the percent of cells infected. SA activates the heme-regulated eIF2α (HRI) kinase, which phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) to induce stress granule (SG) formation. Heat shock (HS), another activator of HRI, also induced eIF2α phosphorylation and SGs in cells. However, HS had no effect on percent infectivity or viral yield but did enhance viral protein expression. These data suggest that SA pre-treatment perturbs the cell in a way that is beneficial for reovirus and that this enhancement is independent of SG induction. Understanding how to manipulate the cellular stress responses during infection to enhance replication could help to maximize the oncolytic potential of reovirus.

Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells.

We describe droplet-assisted RNA targeting by single-cell sequencing (DART-seq), a versatile technology that enables multiplexed amplicon sequencing and transcriptome profiling in single cells. We applied DART-seq to simultaneously characterize the non-A-tailed transcripts of a segmented dsRNA virus and the transcriptome of the infected cell. In addition, we used DART-seq to simultaneously determine the natively paired, variable region heavy and light chain amplicons and the transcriptome of B lymphocytes.

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility.

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO-/- mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO-/- mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO-/- sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO-/- sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO-/- sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.

Conserved Surface Residues on the Feline Calicivirus Capsid Are Essential for Interaction with Its Receptor Feline Junctional Adhesion Molecule A (fJAM-A).

Host cell surface receptors are required for attachment, binding, entry, and infection by nonenveloped viruses. Receptor binding can induce conformational changes in the viral capsid and/or the receptor that couple binding with downstream events in the virus life cycle (intracellular signaling, endocytosis and trafficking, and membrane penetration). Virus-receptor interactions also influence viral spread and pathogenicity. The interaction between feline calicivirus (FCV) and its receptor, feline junctional adhesion molecule A (fJAM-A), on host cells is required for infection and induces irreversible, inactivating conformational changes in the capsid of some viral strains. Cryoelectron microscopy (cryo-EM) structures of FCV bound to fJAM-A showed several possible virus-receptor interactions. However, the specific residues on the viral capsid required for binding are not known. Capsid residues that may be involved in postbinding events have been implicated by isolation of soluble receptor-resistant (srr) mutants in which changes in the capsid protein sequence change the capacity of such srr mutants to be inactivated upon incubation with soluble fJAM-A. To clarify which residues on the surface of FCV are required for its interaction with fJAM-A and to potentially identify residues required for postreceptor binding events, we used the existing atomic-resolution structures of FCV and the FCV-fJAM-A cryo-EM structures to select 14 capsid residues for mutation and preparation of recombinant viral capsids. Using this approach, we identified residues on the FCV capsid that are required for fJAM-A binding and other residues that are not required for binding but are required for infection that are likely important for subsequent postbinding events.IMPORTANCE Feline calicivirus (FCV) is a common cause of mild upper respiratory disease in cats. Some FCV isolates can cause virulent systemic disease. The genetic determinants of virulence for FCV are unknown. We previously found that virulent FCV isolates have faster in vitro growth kinetics than less virulent isolates. Differences in viral growth in vitro may correlate with differences in virulence. Here, we investigated the roles of specific FCV capsid residues on the receptor-virus interaction and viral growth in vitro We show that the capsid protein genes of the virulent FCV-5 isolate determine its faster in vitro growth kinetics compared to those of the nonvirulent FCV-Urbana infectious clone. We also identified residues on the capsid VP1 protein that are important for receptor binding or for steps subsequent to receptor binding. Our data provide further insight into the specific molecular interactions between fJAM-A and the FCV capsid that regulate binding and infectious entry.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.