Fluorescent protein tags affect the condensation properties of a phase-separating viral protein.

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.

Transcriptomic signatures of feline chronic gingivostomatitis are influenced by upregulated IL6.

Feline chronic gingivostomatitis (FCGS) is a relatively common and debilitating disease characterized by bilateral inflammation and ulceration of the caudal oral mucosa, alveolar and buccal mucosa, and varying degrees of periodontal disease. The etiopathogenesis of FCGS remains unresolved. In this study, we performed bulk RNA-seq molecular profiling of affected tissues derived from a cohort of client-owned cats with FCGS compared to tissues from unaffected animals, to identify candidate genes and pathways that can help guide future exploration of novel clinical solutions. We complemented transcriptomic findings with immunohistochemistry and in situ hybridization assays to better understand the biological significance of the results and performed RNA-seq validation of biologically relevant differentially expressed genes using qPCR assays to demonstrate technical reproducibility. Transcriptomic profiles of oral mucosal tissues in cats with FCGS are enriched with immune- and inflammation-related genes and pathways that appear to be largely influenced by IL6, and include NFKB, JAK/STAT, IL-17 and IFN type I and II signaling, offering new opportunities to develop novel clinical applications based on a more rational understanding of the disease.

Transcriptomic signatures of feline chronic gingivostomatitis are influenced by upregulated IL6.

Feline chronic gingivostomatitis (FCGS) is a relatively common and debilitating disease characterized by bilateral inflammation and ulceration of the caudal oral mucosa, alveolar and buccal mucosa, and varying degrees of periodontal disease. The etiopathogenesis of FCGS remains unresolved. In this study, we performed bulk RNA-seq molecular profiling of affected tissues derived from a cohort of client-owned cats with FCGS compared to tissues from unaffected animals, to identify candidate genes and pathways that can help guide future exploration of novel clinical solutions. We complemented transcriptomic findings with immunohistochemistry and in situ hybridization assays to better understand the biological significance of the results and performed RNA-seq validation of selected differentially expressed genes using qPCR assays to demonstrate technical reproducibility. Transcriptomic profiles of oral mucosal tissues in cats with FCGS are enriched with immune- and inflammation-related genes and pathways that appear to be largely influenced by IL6 , and include NFKB, JAK/STAT, IL-17 and IFN type I and II signaling, offering new opportunities to develop novel clinical applications based on a more rational understanding of the disease.

Differences in career paths of alumni of the Cornell Leadership Program from North American universities compared with those alumni from universities in other countries.

OBJECTIVE: To characterize and compare the careers of alumni of the Cornell Leadership Program for Veterinary Students according to the countries where they studied and obtained their veterinary qualification. The Cornell Leadership Program is a 10-week residential research experience program for veterinary students from around the world who have ambitions for research-related careers. SAMPLE: Data on the career development of all 672 alumni were collected each year over the period of 1990 to 2019. PROCEDURES: The annual career profile of each alumnus was recorded and coded in 1 of 28 different categories. The careers and postveterinary qualifications of alumni from universities in the US and Canada (referred to as North American universities) were compared with those alumni who graduated from universities in other countries. RESULTS: Analysis of this 30-year database revealed that a considerable proportion (45.7% [307/672]) of the total 672 alumni are following the traditional career path of veterinary clinical practice rather than the research-related careers they aspired to as students during the Leadership Program. Furthermore, a higher proportion of the 325 North American alumni (56% [182/325]) were in clinical practice compared with 33.6% (112/333) of the 333 alumni from other countries. CLINICAL RELEVANCE: Many veterinary schools now provide research experience programs to encourage highly talented students who have ambitions for careers in which they can advance knowledge about animal disease and contribute to solving the health problems of animals through hypothesis-based research. Comparison of the careers of the Leadership Program alumni indicates that research experience alone is not sufficient to maintain the career goals of alumni. Follow-up mentoring of alumni of such programs is recommended while they complete their veterinary studies to reinforce their career aspirations and provide advice on how to achieve research-related careers.

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.

Curriculum Guidelines for Graduate and Undergraduate Virology Courses.

Curriculum guidelines for virology are needed to best guide student learning due to the continuous and ever-increasing volume of virology information, the need to ensure that undergraduate and graduate students have a foundational understanding of key virology concepts, and the importance in being able to communicate that understanding to both other virologists and nonvirologists. Such guidelines, developed by virology educators and the American Society for Virology Education and Career Development Committee, are described herein.

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.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein µNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus µNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

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.

The Paradoxes of Viral mRNA Translation during Mammalian Orthoreovirus Infection.

De novo viral protein synthesis following entry into host cells is essential for viral replication. As a consequence, viruses have evolved mechanisms to engage the host translational machinery while at the same time avoiding or counteracting host defenses that act to repress translation. Mammalian orthoreoviruses are dsRNA-containing viruses whose mRNAs were used as models for early investigations into the mechanisms that underpin the recognition and engagement of eukaryotic mRNAs by host cell ribosomes. However, there remain many unanswered questions and paradoxes regarding translation of reoviral mRNAs in the context of infection. This review summarizes the current state of knowledge about reovirus translation, identifies key unanswered questions, and proposes possible pathways toward a better understanding of reovirus translation.

Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction.

Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic double-stranded RNA (dsRNA). The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCE We use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here, we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

Tracking Veterinary Students Who Aspire to Careers in Science.

The Cornell Leadership Program for Veterinary Students is an intensive 10-week learning experience intended to guide competitively selected scholars into careers in science and public health. It features independent research, vocational counseling, and student-directed learning modules. Program scholars are encouraged to objectively evaluate graduate training as preparation for careers promoted by the program. Prominence is given to experiential learning through research, participation in program enrichment modules, and inspirational experiences achieved through group meetings and individual interactions with established scientists. Program alumni are monitored to determine how the careers they pursue relate to their earlier-stated ambitions. In addition, subjective assessments are made of the quality of graduate training and its impact on alumni career paths. The influence of mentors, vocational counseling, and inspirational experiences on subsequent training is also subjectively assessed. Information is obtained from students' anonymous responses to questionnaires and recorded interviews. Program alumni are contacted annually to determine their current activities and career aspirations. The Leadership Program encourages program graduates to undertake careers in science and public health, yet an unanticipated number of alumni enter private veterinary practice. A factor relevant to that outcome is that many students destined for practice lack a definitive career plan. Persuading veterinary students to consider careers in research or public service is challenging but worth the effort. Critical to that connection is the need for veterinary students to objectively evaluate graduate training options because the vocations they follow appear to be strongly influenced by the experiences they choose.

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.

A pLOT of Viral Persistence.

In this issue of Cell Host & Microbe, Bouziat et al. (2018) and Van Winkle et al. (2018) find that the capsid gene of murine norovirus (MNV) functions as a trigger of host inflammation. These studies specifically describe how MNV-induced inflammation promotes loss of oral tolerance and persistent viral infection, respectively.

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.

Respiratory disease in ball pythons (Python regius) experimentally infected with ball python nidovirus.

Circumstantial evidence has linked a new group of nidoviruses with respiratory disease in pythons, lizards, and cattle. We conducted experimental infections in ball pythons (Python regius) to test the hypothesis that ball python nidovirus (BPNV) infection results in respiratory disease. Three ball pythons were inoculated orally and intratracheally with cell culture isolated BPNV and two were sham inoculated. Antemortem choanal, oroesophageal, and cloacal swabs and postmortem tissues of infected snakes were positive for viral RNA, protein, and infectious virus by qRT-PCR, immunohistochemistry, western blot and virus isolation. Clinical signs included oral mucosal reddening, abundant mucus secretions, open-mouthed breathing, and anorexia. Histologic lesions included chronic-active mucinous rhinitis, stomatitis, tracheitis, esophagitis and proliferative interstitial pneumonia. Control snakes remained negative and free of clinical signs throughout the experiment. Our findings establish a causal relationship between nidovirus infection and respiratory disease in ball pythons and shed light on disease progression and transmission.

Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.

Vocational choices made by alumni of the Leadership Program for Veterinary Students at Cornell University.

OBJECTIVE To compare vocational aspirations and outcomes of participants in the 10-week Leadership Program for Veterinary Students at Cornell University. DESIGN Survey. SAMPLE Veterinary students who participated in the program between 1990 and 2013. PROCEDURES Questionnaires that sought information about the career aspirations of participants at the beginning and end of the program were reviewed, along with records documenting the career progression of participants, audio recordings of interviews conducted with students, and notes of vocation-oriented counseling sessions held during each year's program. RESULTS At the conclusion of the program, 143 of 174 (82%) participants indicated they were more likely than not to undertake research training after completing their veterinary degree, compared with 106 of 174 (61%) at the beginning. Participation also stimulated interest in residency training and industry, but did little to promote interest in careers in government or the military. The percentage of participants who indicated they were more likely than not to pursue additional training in private practice decreased from 97 of 174 (56%) at the beginning of the program to 75 of 174 (43%) at the end. Information on career progression was available for 391 individuals, of whom 177 (45%) were pursuing careers of the kind envisioned by the program. However, 189 (48%) participants had a career in general or specialty clinical practice. CONCLUSIONS AND CLINICAL RELEVANCE The Leadership Program appeared to have a short-term influence on careers anticipated by program participants. However, a substantial proportion pursued careers in clinical practice after graduation.

Virus-mediated compartmentalization of the host translational machinery.

UNLABELLED: Viruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression. IMPORTANCE: Viruses lack biosynthetic capabilities and depend upon the host for protein synthesis. This dependence requires viruses to evolve mechanisms to coerce the host translational machinery into synthesizing viral proteins in the face of ongoing cellular stress responses that suppress global protein synthesis. Reoviruses replicate and assemble within cytoplasmic inclusions called viral factories. However, synthesis of viral proteins was thought to occur in the cytosol. To identify the site(s) of viral translation, we undertook a microscopy-based approach using ribopuromycylation to detect active translation. Here, we report that active translation occurs within viral factories and that translational factors are compartmentalized within factories. Furthermore, we find that the reovirus nonstructural protein σNS associates with 43S preinitiation complexes at the factory margins, suggesting a role for σNS in translation. Together, virus-induced compartmentalization of the host translational machinery represents a strategy for viruses to spatiotemporally couple viral protein synthesis with viral replication and assembly.