Antiviral immunity triggered by infection-induced host transposable elements

Host silencing of transposable elements (TEs) is critical to prevent genome damage and inappropriate inflammation. However, new evidence suggests that a virus-infected host may re-activate TEs and co-opt them for antiviral defense. RNA-Seq and specialized bioinformatics have revealed the diversity of virus infections that induce TEs. Furthermore, studies with influenza virus have uncovered how infection-triggered changes to the SUMOylation of TRIM28, an epigenetic co-repressor, lead to TE de-repression. Importantly, there is a growing appreciation of how de-repressed TEs stimulate antiviral gene expression, either via cis-acting enhancer functions or via their recognition as viral mimetics by innate immune nucleic acid sensors (e.g. RIG-I, mda-5 and cGAS). Understanding how viruses trigger, and counteract, TE-based antiviral immunity should provide insights into pathogenic mechanisms

Avoiding culture shock with the SARS-CoV-2 spike protein

When culturing SARS-CoV-2 in the laboratory it is vital to avoid deletions in the gene for the spike protein that could affect the interpretation of experiments

SARS-CoV-2 takes the bait: Exosomes as endogenous decoys

Writing in PLOS Biology, Ching and colleagues show that ACE2-decorated exosomes are deployed as natural inhibitory decoys against SARS-CoV-2. High decoy levels correlate with improved patient outcomes, suggesting they directly help COVID-19 recovery and supporting the concept of successful future decoy-based therapies

Ultra-Rare BRD9 Loss-of-Function Variants Limit the Antiviral Action of Interferon

The human type I interferon (IFN) system is central to innate immune defense, and is essential to protect individuals against severe viral disease. Consequently, genetic disruption of IFN signaling or effector mechanisms is extremely rare, as affected individuals typically suffer life-threatening infections at an early age. While loss-of-function (LOF) mutations in canonical JAK-STAT signaling genes (such as IFNAR2, TYK2, STAT1, STAT2 and IRF9) have previously been characterized, little is known about the consequences of mutations in other human factors required for IFN signaling. Here, we studied the impact of rare human genetic variants in the recently identified contributor to IFN-stimulated gene expression and antiviral activity, bromodomain-containing protein 9 (BRD9). Using a cell-based BRD9 knock-out and reconstitution model system, we functionally assessed 12 rare human BRD9 missense variants predicted to impair protein function, as well as 3 ultra-rare human BRD9 LOF variants that lead to truncated versions of BRD9. As compared to wild-type BRD9, none of the 12 BRD9 missense variants affected the ability of exogenous IFN to limit virus replication. In contrast, all 3 truncated BRD9 LOF variants failed to allow exogenous IFN to function efficiently, as evidenced by exacerbated replication of an IFN-sensitive virus and diminished IFN-stimulated gene expression. Thus, while no homozygous BRD9 LOF carriers have yet been identified, our results predict that such extremely rare individuals would exhibit a compromised ability to mount a fully protective IFN-mediated antiviral response. Genetic variation in BRD9 could be considered in future studies to understand the infection susceptibility of some individuals

Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses

A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than parental. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern

Human interactome of the influenza B virus NS1 protein

This work was partially supported by NIAID grant U19AI106754, and by CRIP (Center for Research in Influenza Pathogenesis), an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract #HHSN272201400008C) to A. G-S. Work at the University of Zurich was supported by the Swiss National Science Foundation (grant 31003A_159993 to BGH).NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.Publisher PDFPeer reviewe

An influenza virus-triggered SUMO switch orchestrates co-opted endogenous retroviruses to stimulate host antiviral immunity

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of "non-self" nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of "self" double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.</p

A transient homotypic interaction model for the influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins

SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern