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.

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.

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.

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.

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.

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.

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.).

Ubiquitination Upregulates Influenza Virus Polymerase Function.

The influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation. IMPORTANCE The cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

Phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery.

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle.

Conserved features of the PB2 627 domain impact influenza virus polymerase function and replication.

UNLABELLED: Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE: Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

Influenza A virus polymerase is a site for adaptive changes during experimental evolution in bat cells.

UNLABELLED: The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE: Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity.

Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.

The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.

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.

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.

Fluorescence resonance energy transfer-based intracellular assay for the conformation of hepatitis C virus drug target NS5A.

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests that large portions of the carboxy terminus of NS5A are in an unfolded and disordered state. Quercetin, a natural product known to disrupt NS5A function in cells, specifically disrupted a conformationally specific domain 3 FRET signal. Intermolecular FRET indicated that the NS5A amino termini, but not other regions, are in close proximity in multimeric complexes. Overall, this assay provides a new window on the intracellular conformation(s) of NS5A and how the conformation changes in response to cellular and viral components of the replication and assembly complex as well as antiviral drugs.

Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers.

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.

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.