The COVID-19 pandemic illustrated the need to develop medical countermeasures against emerging infectious diseases. As viruses rely on cellular machinery for replication, host-directed antivirals (HDAs) may complement conventional antiviral strategies in a manner that offers broad-spectrum efficacy, including against novel viruses, and a potentially higher barrier to viral escape. Despite their potential, HDAs are under-represented as therapeutics, partly due to a lack of consensus on druggable host targets. To address this, we have performed a meta-analysis of 62 functional genomics studies involving viral families of pandemic concern, including Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picronaviridae, Poxviridae and Togaviridae. Using a robust rank aggregation and protein-protein interaction network approach, host factors and cellular processes required by multiple virus families were identified, including the V-type ATPase complex, glycosaminoglycan synthesis, Golgi trafficking and endoplasmic reticulum membrane protein insertion. These pathways include those with known relevance to infection by some viruses while providing novel insights into the lifecycles of others. Therapeutic targeting of these top ranked host factors is also discussed, with several already possessing small molecule inhibitors, highlighting their therapeutic potential. Antivirals are an essential component of medical countermeasures against viral disease and fulfil a complementary role to vaccines. Importantly, they may provide the only therapeutic option for pathogens lacking effective vaccines or for individuals unable to be vaccinated. This analysis furthers our understanding of the virus-host interface and nominates cellular targets for the future development of HDAs as medical countermeasures for pandemic resilience.

Viruses exploit cellular machinery to complete their replication cycle. Furthering our understanding of this process provides insight into the mechanism of virus replication and potential targets for antiviral therapeutics. Genome-wide CRISPR screens have identified cellular pathways important in the SARS-COV-2 infection process, including vesicular traffic, lipid homeostasis and PI3K signalling. Functional genomics-driven analysis of host-encoded microRNAs (miRNAs) impacting SARS-CoV-2 infection would provide further unbiased and discovery-driven insight into the host-pathogen interface. Here we present findings from genome-wide complementary miRNA mimic and inhibitor screens performed in a bio-safety level (BSL)-4 laboratory using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines. This dataset has identified both miRNA promoters and inhibitors of SARS-CoV-2 replication which may be used by researchers to further explore therapeutic targets against SARS-CoV-2 and the host factors influencing COVID pathogenesis.

Comprehensive characterization of bovine immune cell populations is essential for improving animal welfare and disease resilience. We performed single-cell RNA sequencing on over 29,000 peripheral blood mononuclear cells (PBMCs) from Angus cattle stratified by delayed-type hypersensitivity (DTH), a proxy for the cellular immune response (Cell-IR). Unsupervised clustering identified major immune populations including CD4⁺ and CD8⁺ T cells, γδ T cells, B cells, monocytes, and dendritic cells. Differential gene expression suggests that low Cell-IR cattle have in elevated NKT inflammatory response, while high Cell-IR cattle have increased CD8- γδ T cell and pro-inflammatory myeloid activity. Intercellular communication analysis using CellChat highlighted pro-inflammatory cytokine cascades, particularly the IL-1β– IL-1R1 ligand-receptor interactions. This study provides a high-resolution atlas of Angus PBMCs and establishes a framework for linking immune cell composition with functional immune phenotypes in cattle.

Inflammation and lipid regulator with UBA-like and NBR1-like domains (ILRUN) is a protein-encoding gene associated with innate immune signaling, lipid metabolism and cancer. In the context of innate immunity, ILRUN inhibits IRF3-mediated transcription of antimicrobial and proinflammatory cytokines by inducing degradation of the transcriptional coactivators CBP and p300. There remains a paucity of information, however, regarding the innate immune roles of ILRUN beyond in vitro analyses. To address this, we utilize a knockout mouse model to investigate the effect of ILRUN on cytokine expression in splenocytes and on the development of immune cell populations in the spleen and thymus. We show elevated production of tumor necrosis factor and interleukin-6 cytokines in ILRUN-deficient splenocytes following stimulation with the innate immune ligands polyinosinic: polycytidylic acid or lipopolysaccharide. Differences were also observed in the populations of several T cell subsets, including regulatory, mucosal-associated invariant and natural killer. These data identify novel functions for ILRUN in the development of certain immune cell populations and support previous in vitro findings that ILRUN negatively regulates the synthesis of pathogen-stimulated cytokines. This establishes the ILRUN knockout mouse model as a valuable resource for further study of the functions of ILRUN in health and disease.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year.

The rabies virus (RABV) phosphoprotein (P protein) is expressed as several isoforms, which differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several sequences, including nuclear localization (NLS) and export (NES) sequences. This appears to underpin a functional diversity enabling multiple functions in viral replication and modulation of host biology. Mechanisms regulating trafficking are poorly defined, but phosphorylation by protein kinase C (PKC) in the P protein C-terminal domain (PCTD) regulates nuclear trafficking, mediated by PCTD-localized NLS/NES sequences, indicating that phosphorylation contributes to functional diversity. The molecular mechanism underlying the effects of PKC, and potential roles in regulating other host-cell interactions are unresolved. Here, we assess effects of phosphorylation on the P3 isoform, which differs from longer isoforms through an ability to localize to the nucleus and associate with MTs, which are associated with antagonism of interferon (IFN) signaling. We find that phosphomimetic mutation of the PKC site S210 inhibits nuclear accumulation and MT association/bundling. Structural analysis indicated that phosphomimetic mutation induces no significant structural change to the NLS/NES but results in the side chain of N226 switching its interactions from E228, within the NES, to E210. Intriguingly, N226 is the sole substituted residue between the PCTD of the pathogenic IFN-resistant RABV strain Nishigahara and a derivative attenuated IFN-sensitive strain Ni-CE, inhibiting P3 nuclear localization and MT association. Thus, S210 phosphorylation appears to impact on N226/E228 to regulate P protein localization, with N226 mutation in Ni-CE mimicking a constitutively phosphorylated state resulting in IFN sensitivity and attenuation. IMPORTANCE Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules. Previous studies showed that phosphorylation of the P protein C-terminal domain (PCTD) at S210, near nuclear trafficking sequences, regulates nucleocytoplasmic localization, indicating key roles in functional diversity. The molecular mechanisms of this regulation have remained unknown. Here, we show that phosphomimetic mutation of S210 regulates nuclear localization and MT association. This regulation does not appear to result from disrupted PCTD structure, but rather from a switch of specific side chain interactions of N226. Intriguingly, N226 was previously implicated in P protein nuclear localization/MT association, immune evasion, and RABV pathogenesis, through undefined mechanisms. Our data indicate that the S210-N226 interface is a key regulator of virus-host interactions, which is significant for pathogenesis.

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host–pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host–pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host–pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease.

Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.

Interferon (IFN)-mediated immunity is a central mode of defense against viral infection and evasion of this immune response is critical to the pathogenicity of viruses. IFN-antagonist proteins have recently been shown to interact with host microtubules (MTs) demonstrating a novel mechanism for subverting the IFN response (1). Using super-resolution fluorescence microscopy we have imaged the association of the IFN-antagonist protein with host cell MTs and the consequent changes to the architecture of the cell cytoskeleton. Super resolution imaging was achieved using the dSTORM approach on a home-built set-up. dSTORM imaging of MT architecture in cells expressing Rhabdovirus proteins has allowed previously unobservable changes caused by the IFN-antagonist proteins to be detected. Transfected cells COS7 cells labeled with Alexa 647 (Figure - left panel) show a high degree of bundling and abnormal curvature of the microtubule skeleton. In contrast, healthy control cells (Figure - center panel) show normal microtubule architecture. (1) K.G. Lieu, A. Brice, L. Wiltzer, B. Hirst, D.A. Jans, D. Blondel and G.W. Moseley, Journal of Virology, (2013) doi: 10/1128/JVI.00989-13.

3044-Pos Board B736 Mouse Retina Imaging by Means of Inverted Selective Plane Illumination Microscopy (ISPIM) Zeno Lavagnino, Francesca Cella Zanacchi, Luca Lanzanò, Alberto Diaspro. Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy. The retina has a very complex structure andworking principle, since its task is to elaborate and transmit light signals to the optical nerve. Investigating its three dimensional structure in details represents a key point to understand its behavior. Lightsheet microscopy has been established as a powerful technique to reveal structures in thick biological specimens. Nevertheless, standard Selective Plane Illumination Microscopes (1) still rely on the embedding of the sample within agar gel or some other aqueous compound. This kind of solution limits the variety of samples that one can investigate under SPIM. To get rid of these limitations, we developed a version of inverted SPIM (2) which takes advantage of light sheet microscopy in a vertical geometry, where the specimen can be placed on standard petri dishes or coverslips, thus enhancing the portfolio of usable samples for SPIM.We performed imaging of transgenicmouse retina expressing standard fluorescent proteins (3), being interested on the retinal ganglion cell layer, to produce a 3D reconstruction of the development of dendrites. We were able to perform volume acquisition covering the entire thickness of the retina (approximately 300 microns) in less than 10 seconds with a large field of view, using a low NA detection objective. This technique combines the advantages of placing the sample in a standard substrate for microscopy with the speed of a widefield microscope, obtaining a resolution (with low NA objective) comparable to confocal microscopy. Moreover, 3D imaging can be achieved deeper into the sample than using standard microscopy techniques (4). (1) Ahrens M.B. et al. Nat. Methods 10 413-420 (2013). (2) Wu Y. et al. PNAS 108 (43) (2011). (3) Renvision project, FP7-ICT-2011-9 (NBIS) Grant Agreement Number 600847. (4) Lavagnino Z. et al. Opt. Express 21 (5) (2013).

Immune evasion by rabies virus depends on targeting of the signal transducers and activator of transcription 1 (STAT1) and STAT2 proteins by the viral interferon antagonist P protein, but targeting of other STAT proteins has not been investigated. Here, we find that P protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human-pathogenic viruses.

Viral interaction with the microtubule (MT) cytoskeleton is critical to infection by many viruses. Most data regarding virus-MT interaction indicate key roles in the subcellular transport of virions/viral genomic material to sites of replication, assembly and egress. However, the MT cytoskeleton orchestrates diverse processes in addition to subcellular cargo transport, including regulation of signaling pathways, cell survival and mitosis, suggesting that viruses, expert manipulators of the host cell, may use the virus-MT interface to control multiple aspects of cell biology. Several lines of evidence support this idea, indicating that specific viral proteins can modify MT dynamics and/or structure and regulate processes such as apoptosis and innate immune signaling through MT-dependent mechanisms. Here, the authors review general aspects of virus-MT interactions, with emphasis on viral mechanisms that modify MT dynamics and functions to affect processes beyond virion transport. The emerging importance of discrete viral protein-MT interactions in pathogenic processes indicates that these interfaces may represent new targets for future therapeutics and vaccine development.

Background: Viral evasion of the interferon (IFN)-mediated innate immune response is essential to pathogenic infection, and depends on the expression of viral IFN-antagonist proteins that interact with and inhibit host factors of the IFN-response, including the transcription factor STAT 1. In vitro studies have suggested that immune evasion by highly pathogenic viruses such as rabies (RABV), the causative agent of >55,000 human fatalities/year, and Nipah virus, involves several IFN-antagonist protein isoforms that mediate distinct IFN-inhibitory mechanisms in specific subcellular compartments. For example, we have shown that the RABV IFN-antagonist isoforms P1 and P3 inhibit IFN-dependent STAT1 signaling by distinct mechanisms dependent on their differential localisation in the cytoplasm and nucleus, and at the microtubule (MT) cytoskeleton. It has thus been hypothesised that the cumulative effect of these mechanisms is required for efficient shut-down of the IFN response. However, the importance of these in vitro observations to viral virulence in vivo is unknown. Methods: To examine the role of different IFN-antagonist isoforms in virulence, we used a combined approach to bridge molecular/cellular approaches with in vivo pathogenicity studies. Using novel quantitative live-cell imaging approaches/high-end confocal laser scanning microscopy, IFN-signalling assays and in vitro protein interaction assays, we characterised the molecular events underlying the differing functions of P1 and P3, including nucleocytoplasmic trafficking, MT-association, and inhibition of STAT1 signaling, and identified mutations that can specifically inhibit these processes. Using reverse genetics, we generated recombinant RABV defective in these functions, and evaluated viral infectivity and immune evasion in vitro, and pathogenicity in infected animals. Results: The study revealed for the first time that mutations causing defects in specific properties of the P1 and P3 isoforms, including nuclear export and MT-association, directly impact their IFN-antagonistic functions, the sensitivity of infectious virus to IFN, and virulence in infected animals. Importantly, the data indicate that the distinct functions of the isoforms make significant but partial contributions to viral pathogenicity in vivo, with full virulence dependent on their combined activity. This data directly support the hypothesis that viruses have evolved multi-pronged, integrated strategies to mediate an effective counterattack against the host immune response. Conclusion: This work contributes significantly to our understanding of the molecular events at the virus-host interface, and their importance to viral evasion of innate immunity, and viral pathogenicity. This is significant not only to RABV but also to other lethal human pathogens, with implications for the development of new vaccines/antivirals.