Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

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.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

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.

Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection.

Host:pathogen interactions dictate the outcome of infection, yet the limitations of current approaches leave large regions of this interface unexplored. Here, we develop a novel fitness-based screen that queries factors important during the middle to late stages of infection. This is achieved by engineering influenza virus to direct the screen by programming dCas9 to modulate host gene expression. Our genome-wide screen for pro-viral factors identifies the cytoplasmic DNA exonuclease TREX1. TREX1 degrades cytoplasmic DNA to prevent inappropriate innate immune activation by self-DNA. We reveal that this same process aids influenza virus replication. Infection triggers release of mitochondrial DNA into the cytoplasm, activating antiviral signaling via cGAS and STING. TREX1 metabolizes the DNA, preventing its sensing. Collectively, these data show that self-DNA is deployed to amplify innate immunity, a process tempered by TREX1. Moreover, they demonstrate the power and generality of pathogen-driven fitness-based screens to pinpoint key host regulators of infection.

Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.

Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.

Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection.

Intracellular pathogens interact with host factors, exploiting those that enhance replication while countering those that suppress it. Genetic screens have begun to define the host:pathogen interface and establish a mechanistic basis for host-directed therapies. Yet, limitations of current approaches leave large regions of this interface unexplored. To uncover host factors with pro-pathogen functions, we developed a novel fitness-based screen that queries factors important during the middle-to-late stages of infection. This was achieved by engineering influenza virus to direct the screen by programing dCas9 to modulate host gene expression. A genome-wide screen identified the cytoplasmic DNA exonuclease TREX1 as a potent pro-viral factor. TREX1 normally degrades cytoplasmic DNA to prevent inappropriate innate immune activation by self DNA. Our mechanistic studies revealed that this same process functions during influenza virus infection to enhance replication. Infection triggered release of mitochondrial DNA into the cytoplasm, activating antiviral signaling via cGAS and STING. TREX1 metabolized the mitochondrial DNA preventing its sensing. Collectively, these data show that self-DNA is deployed to amplify host innate sensing during RNA virus infection, a process tempered by TREX1. Moreover, they demonstrate the power and generality of pathogen driven fitness-based screens to pinpoint key host regulators of intracellular pathogens.

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

Retasking of canonical antiviral factors into proviral effectors.

Under constant barrage by viruses, hosts have evolved a plethora of antiviral effectors and defense mechanisms. To survive, viruses must adapt to evade or subvert these defenses while still capturing cellular resources to fuel their replication cycles. Large-scale studies of the antiviral activities of cellular proteins and processes have shown that different viruses are controlled by distinct subsets of antiviral genes. The remaining antiviral genes are either ineffective in controlling infection, or in some cases, actually promote infection. In these cases, classically defined antiviral factors are retasked by viruses to enhance viral replication. This creates a more nuanced picture revealing the contextual nature of antiviral activity. The same protein can exert different effects on replication, depending on multiple factors, including the host, the target cells, and the specific virus infecting it. Here, we review numerous examples of viruses hijacking canonically antiviral proteins and retasking them for proviral purposes.

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.

Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks.

Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The bottlenecks within hosts and their potential impacts on evolutionary pathways taken during infection are poorly understood. To address this, we created highly diverse IAV libraries bearing molecular barcodes on two gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in "islands" of infection in lung lobes, each with genetically distinct populations. We perform site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and track viral diversity as infection spreads to the trachea and lungs. We detect extensive compartmentalization of discrete populations within lung lobes. Bottleneck events and localized replication stochastically sample individual viruses from the upper respiratory tract or the trachea that become the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with limited evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity.

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase.

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

Influenza virus repurposes the antiviral protein IFIT2 to promote translation of viral mRNAs.

Cells infected by influenza virus mount a large-scale antiviral response and most cells ultimately initiate cell-death pathways in an attempt to suppress viral replication. We performed a CRISPR-Cas9-knockout selection designed to identify host factors required for replication after viral entry. We identified a large class of presumptive antiviral factors that unexpectedly act as important proviral enhancers during influenza virus infection. One of these, IFIT2, is an interferon-stimulated gene with well-established antiviral activity but limited mechanistic understanding. As opposed to suppressing infection, we show in the present study that IFIT2 is instead repurposed by influenza virus to promote viral gene expression. CLIP-seq demonstrated that IFIT2 binds directly to viral and cellular messenger RNAs in AU-rich regions, with bound cellular transcripts enriched in interferon-stimulated mRNAs. Polysome and ribosome profiling revealed that IFIT2 prevents ribosome pausing on bound mRNAs. Together, the data link IFIT2 binding to enhanced translational efficiency for viral and cellular mRNAs and ultimately viral replication. Our findings establish a model for the normal function of IFIT2 as a protein that increases translation of cellular mRNAs to support antiviral responses and explain how influenza virus uses this same activity to redirect a classically antiviral protein into a proviral effector.

Post-Translation Regulation of Influenza Virus Replication.

Influenza virus exploits cellular factors to complete each step of viral replication. Yet, multiple host proteins actively block replication. Consequently, infection success depends on the relative speed and efficacy at which both the virus and host use their respective effectors. Post-translational modifications (PTMs) afford both the virus and the host means to readily adapt protein function without the need for new protein production. Here we use influenza virus to address concepts common to all viruses, reviewing how PTMs facilitate and thwart each step of the replication cycle. We also discuss advancements in proteomic methods that better characterize PTMs. Although some effectors and PTMs have clear pro- or antiviral functions, PTMs generally play regulatory roles to tune protein functions, levels, and localization. Synthesis of our current understanding reveals complex regulatory schemes where the effects of PTMs are time and context dependent as the virus and host battle to control infection.

Experimental Approaches to Identify Host Factors Important for Influenza Virus.

An ever-expanding toolkit of experimental methods provides the means to discover and characterize host factors important for influenza virus. Here, we describe common methods for investigating genetic relationships and physical interactions between virus and host. A comprehensive knowledge of host:virus interactions is key to understanding how influenza virus exploits the host cell and to potentially identify vulnerabilities that may be manipulated to prevent or treat disease.

EPS8 Facilitates Uncoating of Influenza A Virus.

All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We use gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identifies the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirm its importance in enhancing FLUAV infection and titers. Loss of EPS8 does not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associates with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes is significantly delayed in the absence of EPS8. Our study identifies EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.