Understanding the neurological implications of acute and long COVID using brain organoids.

As early as in the acute phase of the coronavirus disease 2019 (COVID-19) pandemic, the research community voiced concerns about the long-term implications of infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like many other viruses, can trigger chronic disorders that last months or even years. Long COVID, the chronic and persistent disorder lasting more than 12 weeks after the primary infection with SARS-CoV-2, involves a variable number of neurological manifestations, ranging from mild to severe and even fatal. In vitro and in vivo modeling suggest that SARS-CoV-2 infection drives changes within neurons, glia and the brain vasculature. In this Review, we summarize the current understanding of the neuropathology of acute and long COVID, with particular emphasis on the knowledge derived from brain organoid models. We highlight the advantages and main limitations of brain organoids, leveraging their human-derived origin, their similarity in cellular and tissue architecture to human tissues, and their potential to decipher the pathophysiology of long COVID.

Role of Siglecs in viral infections: A double-edged sword interaction.

Sialic-acid-binding immunoglobulin-like lectins are cell surface immune receptors known as Siglecs that play a paramount role as modulators of immunity. In recent years, research has underscored how the underlaying biology of this family of receptors influences the outcome of viral infections. While Siglecs are needed to promote effective antiviral immune responses, they can also pave the way to viral dissemination within tissues. Here, we review how recent preclinical findings focusing on the interplay between Siglecs and viruses may translate into promising broad-spectrum therapeutic interventions or key biomarkers to monitor the course of viral infections.

Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections.

Mutations in the H7 HA and PB1 genes of avian influenza a viruses increase viral pathogenicity and contact transmission in guinea pigs.

Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Alternative interaction sites in the influenza A virus nucleoprotein mediate viral escape from the importin-α7 mediated nuclear import pathway.

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin-α7 is a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Here, we elucidate the mechanisms by which influenza A viruses may escape replicative restriction in the absence of importin-α7. To address this question, we assessed viral evolution in mice lacking the importin-α7 gene. We show that three mutations in particular occur with high frequency in the viral nucleoprotein (NP) protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-α7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin-α7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin-α7 to replicative fitness in human cells.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors.

Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation ('priming state') and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the 'apo' state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the 'apo' state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive 'apo' state.

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method.

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

Pregnancy-Related Immune Adaptation Promotes the Emergence of Highly Virulent H1N1 Influenza Virus Strains in Allogenically Pregnant Mice.

Pregnant women are at high risk for severe influenza disease outcomes, yet insights into the underlying mechanisms are limited. Here, we present models of H1N1 infection in syngenic and allogenic pregnant mice; infection in the latter mirrors the severe course of 2009 pandemic influenza in pregnant women. We found that the anti-viral immune response in the pregnant host was significantly restricted as compared to the non-pregnant host. This included a reduced type I interferon response as well as impaired migration of CD8+ T cells into the lung. The multi-faceted failure to mount an anti-viral response in allogenic pregnant mice resulted in a less stringent selective environment that promoted the emergence of 2009 H1N1 virus variants that specifically counteract type I interferon response and mediate increased viral pathogenicity. These insights underscore the importance of influenza vaccination compliance in pregnant women and may open novel therapeutic avenues.

H7N9 Influenza A Virus Exhibits Importin-α7-Mediated Replication in the Mammalian Respiratory Tract.

The acute respiratory distress syndrome (ARDS) is the leading cause of death in influenza A virus (IAV)-infected patients. Hereby, the cellular importin-α7 gene plays a major role. It promotes viral replication in the lung, thereby increasing the risk for the development of pneumonia complicated by ARDS. Herein, we analyzed whether the recently emerged H7N9 avian IAV has already adapted to human importin-α7 use, which is associated with high-level virus replication in the mammalian lung. Using a cell-based viral polymerase activity assay, we could detect a decreased H7N9 IAV polymerase activity when importin-α7 was silenced by siRNA. Moreover, virus replication was diminished in the murine cells lacking the importin-α7 gene. Consistently, importin-α7 knockout mice presented reduced pulmonary virus titers and lung lesions as well as enhanced survival rates compared to wild-type mice. In summary, our results show that H7N9 IAV have acquired distinct features of adaptation to human host factors that enable enhanced virulence in mammals. In particular, adaptation to human importin-α7 mediates elevated virus replication in the mammalian lung, which might have contributed to ARDS observed in H7N9-infected patients.

Structural insights into RNA synthesis by the influenza virus transcription-replication machine.

Influenza virus is a segmented, negative strand RNA virus with each genome segment being packaged in a distinct ribonucleoprotein particle (RNP). The RNP consists of the heterotrimeric viral RNA-dependent RNA polymerase bound to the conserved 5' and 3' ends of the genome segment (the viral promoter) with the rest of the viral RNA (vRNA) being covered by multiple copies of nucleoprotein. This review focusses on the new insights that recent crystal structures have given into the detailed molecular mechanisms by which the polymerase performs both transcription and replication of the vRNA genome. Promoter binding, in particular that of 5' end, is essential to allosterically activate all polymerase functions. Transcription is initiated by the hijacking of nascent, capped host transcripts by the process of 'cap-snatching', for which the viral polymerase makes an essential interaction with the C-terminal domain (CTD) of cellular RNA polymerase II. The structures allow a coherent mechanistic model of the subsequent cap-snatching, cap-dependent priming, elongation and self-polyadenylation steps of viral mRNA synthesis. During replication, the vRNA is copied without modification into complementary RNA (cRNA) which is packaged into cRNPs. A priming loop located in the polymerase active site is required for the unprimed synthesis of cRNA from vRNA, but is not required for cRNA to vRNA replication due to differences in the mode of initiation of RNA synthesis. Overall a picture emerges of influenza polymerase being a highly complex, flexible and dynamic machine. The challenge remains to understand in more detail how it functions within the RNP and how interacting host factors modulate its activity in the cellular context. Finally, these detailed insights have opened up new opportunities for structure-based antiviral drug design targeting multiple aspects of polymerase function.

Structural basis of an essential interaction between influenza polymerase and Pol II CTD.

The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5' and 3' termini (the 'promoter') of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus. To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II), which enables it to take 5'-capped primers from nascent Pol II transcripts. Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity. We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.

Targeting Importin-α7 as a Therapeutic Approach against Pandemic Influenza Viruses.

Viral drug resistance is believed to be less likely to occur if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely, importin-α7, as a cellular drug target to combat pandemic influenza viruses. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-α7⁻/⁻ mice that otherwise are resistant. Viral escape from importin-α7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-α7⁻/⁻ mouse-adapted strain became even more virulent for wild-type mice than the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways, eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multitarget approach to reduce viral drug resistance. IMPORTANCE Here, we investigated the long-standing hypothesis based on in vitro studies that viral drug resistance occurrence is less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing, in an in vivo animal model, the feasibility of targeting the cellular factor importin-α7, which is crucial for human influenza virus replication and pathogenesis, as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo, and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multitarget approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein.

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2∆ induces the expression of IFNß and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.

Importin-α7 is required for enhanced influenza A virus replication in the alveolar epithelium and severe lung damage in mice.

Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-α7 in H1N1 influenza virus pulmonary tropism by using various ex vivo imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-α7 gene-deficient (α7(-/-)) mice with a recombinant H1N1 influenza virus and compared the in vivo viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in α7(-/-) mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and α7(-/-) mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-α7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice. Importance: Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-α7, for efficient virus replication in the alveolar epithelium of mice. Using complementary ex vivo imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-α7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-α7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.

The nuclear import machinery is a determinant of influenza virus host adaptation.

After viral entry into the cell, the nuclear envelope poses a major cellular barrier that needs to be overcome upon adaptation of highly pathogenic avian influenza viruses (HPAIV) to the new host. To ensure efficient viral transcription and replication in the nucleus of the host cell, the viral polymerase complex of avian influenza viruses needs to switch from recognition of avian to mammalian components of the nuclear import machinery. Recent evidence suggests that influenza viruses have evolved different mechanisms to utilize importin-α isoforms as components of this machinery, bridging pre- and post-nuclear import on both sides of the nuclear envelope.

The influenza virus RNA synthesis machine: advances in its structure and function.

The influenza A viruses are the causative agents of respiratory disease that occurs as yearly epidemics and occasional pandemics. These viruses are endemic in wild avian species and can sometimes break the species barrier to infect and generate new virus lineages in humans. The influenza A virus genome consists of eight single-stranded, negative-polarity RNAs that form ribonucleoprotein complexes by association to the RNA polymerase and the nucleoprotein. In this review we focus on the structure of this RNA-synthesis machines and the included RNA polymerase, and on the mechanisms by which they express their genetic information as mRNAs and generate progeny ribonucleoproteins that will become incorporated into new infectious virions. New structural, biochemical and genetic data are rapidly accumulating in this very active area of research. We discuss these results and attempt to integrate the information into structural and functional models that may help the design of new experiments and further our knowledge on virus RNA replication and gene expression. This interplay between structural and functional data will eventually provide new targets for controlled attenuation or antiviral therapy.

Structural and functional characterization of an influenza virus RNA polymerase-genomic RNA complex.

The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negative-polarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.

The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

The structural basis for cap binding by influenza virus polymerase subunit PB2.

Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.