Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics.Vertebrates have evolved sophisticated innate and adaptive mechanisms of immunity to combat microbial pathogens 1 . In response, viruses and pathogenic bacteria have acquired virulence factors that subvert or disarm host defenses 1 . For example, cellular membranes provide a simple barrier to infection, but viruses such as influenza virus have evolved specific proteins that fuse with membranes to allow viral replication inside host cells 2 . Alternatively, intracellular bacterial pathogens are taken up by phagocytic cells, but they then remodel cellular membranes to prevent their own degradation inside lysosomal compartments 3 . Cellular membranes are key interfaces for host resistance and prime targets for microbial virulence factors. We therefore performed large-scale profiling of palmitoylated proteins in phagocytic cells to identify membrane-associated proteins that contribute to immunity against microbial pathogens. We found that S-palmitoylation of interferon-induced transmembrane protein 3 (IFITM3) enhances its clustering in membranes * Correspondence and requests for materials should be addressed to H.C.H. hhang@rockefeller.edu. Author contributionsJ.S.Y. conceived the study, designed and performed experiments, interpreted data and co-wrote the manuscript; Y.-Y.Y. and G.C. synthesized reagents for palmitoylome profiling studies; B.M., C.B.L. and T.M.M. provided reagents and expertise on influenza virus infections; H.C.H. conceived the study, designed experiments, interpreted data and co-wrote the manuscript. Competing financial interestsThe authors declare no competing financial interests. Additional information RESULTS Proteomic analysis of palmitoylated proteins in DC2.4 cellsTo identify lipid-modified and membrane-associated proteins that may contribute to immune responses to microbial infections, we performed large-scale profiling of fatty-acylated proteins in the mouse DC line DC2.4 (ref. ...
Summary Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly-deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.
Highlights d Replicating influenza A virus (IAV) produces Z-RNAs d IAV Z-RNAs are sensed by host ZBP1 in the nucleus d ZBP1 activates MLKL in the nucleus, triggering nuclear envelope rupture d MLKL-induced nuclear rupture and necroptosis drive IAV disease severity Authors
Viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. We are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. This Review summarizes current knowledge of the types of defective viral genomes generated during the replication of RNA viruses and the functions that they carry out. We highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. Review ARticleNATuRe MicRoBiology extinction interfere with replication of a wild-type viral genome 29,30 . Hypermutations and mutations leading to frame shifts also result in defective viruses 31 (Fig. 1a). One example is mutations introduced by the cellular protein APOBEC3G into pro-retroviruses 32 . APOBEC3G deaminates deoxycytidine nucleotides in the viral DNA, leading to hypermutations that interfere with the ability of the viral genome to integrate into the host genome and replicate. Interestingly, in opposition to an interfering activity for these mutated pro-viruses, it has been proposed that defective proviruses enhance fitness and that complementation among them drives viral persistence and pathogenesis 33,34 .Deletions. The genomic viral species most commonly referred to as DVGs are truncated viral genomes resulting from large internal deletions occurring during viral replication. Deletion DVGs tend to bear single large truncations that remove several or all essential genes required for self-propagation while retaining 5′ and 3′ ends as well as other RNA structural elements required for polymerase binding, replication and/or packaging [35][36][37] . Multiple variants also exist, including DVGs that can be described as 'mosaic' in that they appear to be the result of multiple recombination and rearrangement events, including deletions, insertions, duplications and even inversions of parts of the genome [38][39][40] (Fig. 1b). Copy-backs.Copy-back and snap-back DVGs are rearranged genomes in which a sequence is duplicated in reverse complement to create theoretical stem-like structures (panhandle structures for copy-back DVGs or hairpin structures for snap-back DVGs) 41-43 . Copy-back DVGs have been reported in many negative-sense (ns) RNA viruses and are generated from the 5′ end of the genome in a process that produces DVGs with complementary 3′ and 5′ termini 44,45 . If the complementary region of the DVG comprises almost the entire sequence, the DVG can be further characterized as a snap-back DVG 43 (Fig. 1c). Copy-back DVGs are predicted to be generated when the viral RNA-dependent RNA polymerase (RdRP) detaches from the template and reattaches to the nascent strand, copying back the end of the genome 42,46 .
Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205–targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-γ– and interleukin 2–producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-γ–dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.
The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.
Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.
Dendritic cell (DC) maturation is a crucial event in the development of adaptive immune responses that confer long-lasting protection against reinfection with the same virus. Sendai virus strain Cantell has a particularly strong ability to mature DCs independently of type I IFNs and TLR signaling, currently the best-described pathways for the induction of DC maturation. In this study, we demonstrate that defective-interfering (DI) particles present in Sendai virus-Cantell stocks are required for its robust DC maturation ability. DI particles contain incomplete genomes that are unable to replicate unless the viral polymerase is supplied by coinfection with complete virus. Accordingly, the improvement in the virus-induced maturation of DCs provided by DI particles requires standard virus coinfection and likely results from increased production of dsRNA replication intermediaries. This unique ability of DI particles to stimulate DC maturation cannot be mimicked by simply increasing the dose of standard virus. Furthermore, viruses with weak DC maturation abilities can be converted into potent DC stimulators with the addition of DI particles, supporting a potential application for DI particles as a novel natural adjuvant for viral immunizations.
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