BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Superinfection exclusion creates spatially distinct influenza virus populations.

Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

The SARS-CoV-2 Spike Protein Mutation Explorer: using an interactive application to improve the public understanding of SARS-CoV-2 variants of concern.

Due to the COVID-19 pandemic the virus responsible, SARS-CoV-2, became a source of intense interest for non-expert audiences. The viral spike protein gained particular public interest as the main target for protective immune responses, including those elicited by vaccines. The rapid evolution of SARS-CoV-2 resulted in variations in the spike that enhanced transmissibility or weakened vaccine protection. This created new variants of concern (VOCs). The emergence of VOCs was studied using viral sequence data which was shared through portals such as the online Mutation Explorer of the COVID-19 Genomics UK consortium (COG-UK/ME). This was designed for an expert audience, but the information it contained could be of general interest if suitably communicated. Visualisations, interactivity and animation can improve engagement and understanding of molecular biology topics, and so we developed a graphical educational resource, the SARS-CoV-2 Spike Protein Mutation Explorer (SSPME), which used interactive 3D molecular models and animations to explain the molecular biology underpinning VOCs. User testing showed that the SSPME had better usability and improved participant knowledge confidence and knowledge acquisition compared to COG-UK/ME. This demonstrates how interactive visualisations can be used for effective molecular biology communication, as well as improving the public understanding of SARS-CoV-2 VOCs.

Visualising Viruses.

Viruses pose a challenge to our imaginations. They exert a highly visible influence on the world in which we live, but operate at scales we cannot directly perceive and without a clear separation between their own biology and that of their hosts. Communication about viruses is therefore typically grounded in mental images of virus particles. Virus particles, as the infectious stage of the viral replication cycle, can be used to explain many directly observable properties of transmission, infection and immunity. In addition, their often striking beauty can stimulate further interest in virology. The structures of some virus particles have been determined experimentally in great detail, but for many important viruses a detailed description of the virus particle is lacking. This can be because they are challenging to describe with a single experimental method, or simply because of a lack of data. In these cases, methods from medical illustration can be applied to produce detailed visualisations of virus particles which integrate information from multiple sources. Here, we demonstrate how this approach was used to visualise the highly variable virus particles of influenza A viruses and, in the early months of the COVID-19 pandemic, the virus particles of the then newly characterised and poorly described SARS-CoV-2. We show how constructing integrative illustrations of virus particles can challenge our thinking about the biology of viruses, as well as providing tools for science communication, and we provide a set of science communication resources to help visualise two viruses whose effects are extremely apparent to all of us.

Using Molecular Visualisation Techniques to Explain the Molecular Biology of SARS-CoV-2 Spike Protein Mutations to a General Audience.

Since the COVID-19 pandemic started in 2019, the virus responsible for the outbreak-SARS-CoV-2-has continued to evolve. Mutations of the virus' spike protein, the main protein driving infectivity and transmissibility, are especially concerning as they may allow the virus to improve its infectivity, transmissibility, and ability to evade the immune system. Understanding how specific molecular changes can alter the behaviour of a virus is challenging for non-experts, but this information helps us to understand the pandemic we are living through and the public health measures and interventions needed to bring it under control. In response to communication challenges arising from the COVID-19 pandemic, we recently developed an online educational application to explain the molecular biology of SARS-CoV-2 spike protein mutations to the general public. We used visualisation techniques such as 3D modelling and animation, which have been shown to be highly effective teaching tools in molecular biology, allowing the viewer to better understand protein structure, function, and dynamics. We also included interactive elements for users to learn actively by engaging with the digital content, and consequently improve information retention.This chapter presents the methodological and technological framework which we used to create this resource, the 'SARS-CoV-2 Spike Protein Mutation Explorer' (SSPME). It explains how molecular visualisation and 3D modelling software were used to develop accurate models of relevant proteins; how 3D animation software was used to accurately visualise the dynamic molecular processes of SARS-CoV-2 infection, transmission, and antibody evasion; and how game development software was used to compile the 3D models and animations into a comprehensive, informative interactive application on SARS-CoV-2 spike protein mutations. This chapter indicates how cutting-edge visualisation techniques and technologies can be used to improve science communication about complex topics in molecular biology and infection biology to the general public, something that is critical to gaining control of the continuing COVID-19 pandemic.

TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing.

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

Filamentous influenza viruses.

Clinical isolates of influenza virus produce pleomorphic virus particles, including extremely long filamentous virions. In contrast, strains of influenza that have adapted to laboratory growth typically produce only spherical virions. As a result, the filamentous phenotype has been overlooked in most influenza virus research. Recent advances in imaging and improved animal models have highlighted the distinct structure and functional relevance of filamentous virions. In this review we summarize what is currently known about these strikingly elongated virus particles and discuss their possible roles in clinical infections.

Regulation of influenza A virus nucleoprotein oligomerization by phosphorylation.

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.

Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication.

UNLABELLED: The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP. IMPORTANCE: Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry.

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses--to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data.

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Multiplexed Biosensing of Proteins and Virions with Disposable Plasmonic Assays.

Our growing ability to tailor healthcare to the needs of individuals has the potential to transform clinical treatment. However, the measurement of multiple biomarkers to inform clinical decisions requires rapid, effective, and affordable diagnostics. Chronic diseases and rapidly evolving pathogens in a larger population have also escalated the need for improved diagnostic capabilities. Current chemical diagnostics are often performed in centralized facilities and are still dependent on multiple steps, molecular labeling, and detailed analysis, causing the result turnaround time to be over hours and days. Rapid diagnostic kits based on lateral flow devices can return results quickly but are only capable of detecting a handful of pathogens or markers. Herein, we present the use of disposable plasmonics with chiroptical nanostructures as a platform for low-cost, label-free optical biosensing with multiplexing and without the need for flow systems often required in current optical biosensors. We showcase the detection of SARS-CoV-2 in complex media as well as an assay for the Norovirus and Zika virus as an early developmental milestone toward high-throughput, single-step diagnostic kits for differential diagnosis of multiple respiratory viruses and any other emerging diagnostic needs. Diagnostics based on this platform, which we term "disposable plasmonics assays," would be suitable for low-cost screening of multiple pathogens or biomarkers in a near-point-of-care setting.

Respiratory viruses: New frontiers-a Keystone Symposia report.

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS-CoV-2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever-evolving viruses that develop resistance, leaving therapy efficacy either short-lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Respiratory Viruses: New Frontiers." Researchers presented new insights into viral biology and virus-host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5.

The influenza A virus RNA polymerase is a heterotrimer that transcribes and replicates the viral genome in the cell nucleus. Newly synthesized RNA polymerase subunits must therefore be imported into the nucleus during an infection. While various models have been proposed for this process, the consensus is that the polymerase basic protein PB1 and polymerase acidic protein PA subunits form a dimer in the cytoplasm and are transported into the nucleus by the beta-importin Ran-binding protein 5 (RanBP5), with the PB2 subunit imported separately to complete the trimeric complex. In this study, we characterized the interaction of PB1 with RanBP5 further and assessed its importance for viral growth. In particular, we found that the N-terminal region of PB1 mediates its binding to RanBP5 and that basic residues in a nuclear localization signal are required for RanBP5 binding. Mutating these basic residues to alanines does not prevent PB1 forming a dimer with PA, but does reduce RanBP5 binding. RanBP5-binding mutations reduce, though do not entirely prevent, the nuclear accumulation of PB1. Furthermore, mutations affecting RanBP5 binding are incompatible with or severely attenuate viral growth, providing further support for a key role for RanBP5 in the influenza A virus life cycle.

Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection.

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.