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Influenza viruses are respiratory pathogens that represent a significant threat to public health, despite the large-scale implementation of vaccination programs. It is necessary to understand the detailed and complex interactions between influenza virus and its host cells in order to identify successful strategies for therapeutic intervention. During viral entry, the cellular microenvironment presents invading pathogens with a series of obstacles that must be overcome to infect permissive cells. Influenza hijacks numerous host cell proteins and associated biological pathways during its journey into the cell, responding to environmental cues in order to successfully replicate. The cellular cytoskeleton and its constituent microtubules represent a heavily exploited network during viral infection. Cytoskeletal filaments provide a dynamic scaffold for subcellular viral trafficking, as well as virus-host interactions with cellular machineries that are essential for efficient uncoating, replication, and egress. In addition, influenza virus infection results in structural changes in the microtubule network, which itself has consequences for viral replication. Microtubules, their functional roles in normal cell biology, and their exploitation by influenza viruses will be the focus of this review.

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus–host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral–host interplay at differing levels of complexity—in individual hosts, organs, tissues and cells—and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid–liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.

Influenza A virus (IAV) represents a worldwide threat to public health by causing severe morbidity and mortality every year. Due to high mutation rate, new strains of IAV emerge frequently. These IAVs are often drug-resistant and require vaccine reformulation. A promising approach to circumvent this problem is to target host cell determinants crucial for IAV infection, but dispensable for the cell. Several RNAi-based screens have identified about one thousand cellular factors that promote IAV infection. However, systematic analyses to determine their specific functions are lacking. To address this issue, we developed quantitative, imaging-based assays to dissect seven consecutive steps in the early phases of IAV infection in tissue culture cells. The entry steps for which we developed the assays were: virus binding to the cell membrane, endocytosis, exposure to low pH in endocytic vacuoles, acid-activated fusion of viral envelope with the vacuolar membrane, nucleocapsid uncoating in the cytosol, nuclear import of viral ribonucleoproteins, and expression of the viral nucleoprotein. We adapted the assays to automated microscopy and optimized them for high-content screening. To quantify the image data, we performed both single and multi-parametric analyses, in combination with machine learning. By time-course experiments, we determined the optimal time points for each assay. Our quality control experiments showed that the assays were sufficiently robust for high-content analysis. The methods we describe in this study provide a powerful high-throughput platform to understand the host cell processes, which can eventually lead to the discovery of novel anti-pathogen strategies.

During cell entry, capsids of incoming influenza A viruses (IAVs) must be uncoated before viral ribonucleoproteins (vRNPs) can enter the nucleus for replication. After hemagglutinin-mediated membrane fusion in late endocytic vacuoles, the vRNPs and the matrix proteins dissociate from each other and disperse within the cytosol. Here, we found that for capsid disassembly, IAV takes advantage of the host cell’s aggresome formation and disassembly machinery. The capsids mimicked misfolded protein aggregates by carrying unanchored ubiquitin chains that activated a histone deacetylase 6 (HDAC6)–dependent pathway. The ubiquitin-binding domain was essential for recruitment of HDAC6 to viral fusion sites and for efficient uncoating and infection. That other components of the aggresome processing machinery, including dynein, dynactin, and myosin II, were also required suggested that physical forces generated by microtubule- and actin-associated motors are essential for IAV entry.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein

Influenza is a zoonotic respiratory disease of major public health interest due to its pandemic potential, and a threat to animals and the human population. The influenza A virus genome consists of eight single-stranded RNA segments sequestered within a protein capsid and a lipid bilayer envelope. During host cell entry, cellular cues contribute to viral conformational changes that promote critical events such as fusion with late endosomes, capsid uncoating and viral genome release into the cytosol. In this focused review, we concisely describe the virus infection cycle and highlight the recent findings of host cell pathways and cytosolic proteins that assist influenza uncoating during host cell entry.

Biological processes are inherently continuous, and the chance of phenotypic discovery is significantly restricted by discretising them. Using multi-parametric active regression we introduce the Regression Plane (RP), a user-friendly discovery tool enabling class-free phenotypic supervised machine learning, to describe and explore biological data in a continuous manner. First, we compare traditional classification with regression in a simulated experimental setup. Second, we use our framework to identify genes involved in regulating triglyceride levels in human cells. Subsequently, we analyse a time-lapse dataset on mitosis to demonstrate that the proposed methodology is capable of modelling complex processes at infinite resolution. Finally, we show that hemocyte differentiation in Drosophila melanogaster has continuous characteristics. High-content screening prompted the development of software enabling discrete phenotypic analysis of single cells. Here, the authors show that supervised continuous machine learning can drive novel discoveries in diverse imaging experiments and present the Regression Plane module of Advanced Cell Classifier.

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu. Technologies for identifying receptor-ligand pairs on living cells at physiological conditions remain scarce. Here, the authors develop a mass spectrometry-based ligand receptor capture technology that can identify receptors for a diverse range of ligands at physiological pH with as few as a million cells.

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus–host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref. 1 ). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-β family proteins, binds to a PY-NLS 2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/β and enter the nucleus. Thus, influenza A virus uses dual importin-βs for distinct steps in host cell entry. A member of the importin-β protein family, transportin 1, binds to influenza A virus matrix protein M1 and promotes its removal from the viral ribonucleoproteins (vRNPs), enabling disassembly of vRNP bundles, vRNP interaction with importin-α/β and entry into the nucleus.

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. Individual cells can move material, collectively referred to as cargo, from the outside environment into the cell interior via a process known as endocytosis. The cell then has different routes to transport the packages of cargo, called endocytic vesicles, to specific locations within the cell. Protein-based molecular machines move the cargo and control how it is selected and targeted to different destinations. For example, a molecular machine that contains a protein called CUL3 labels other components of the system with a chemical tag to regulate the route cargo takes in mammalian cells. However, it was not clear how CUL3 can selectively attach the chemical labels. Gschweitl, Ulbricht et al. have now found that another protein called SPOPL provides selectivity for the CUL3-based machine during endocytosis in human cells. The experiments show that SPOPL attaches to endocytic vesicles, and that CUL3 and SPOPL work together to label a specific component of these vesicles called EPS15. The label changes how EPS15 interacts with other proteins. When SPOPL is not present in a cell, EPS15 is unnaturally stable and occupies many of the routes used by endocytic cargos. The cargo directly interacting with EPS15 is then routed on the fast lane to its destination, while other cargo accumulate in a kind of molecular traffic jam. Other proteins like SPOPL are specific for the endocytic system. Exchange of SPOPL with these similar proteins in the CUL3 machine is likely to chemically label a different set of endocytic proteins. Gschweitl, Ulbricht et al.’s next challenge is to identify the selectivity, targeting and coordination of these exchangeable components in the endocytic system.