cGAS is activated by DNA in a length‐dependent manner

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP‐AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length‐dependent manner. This is observed over a wide length‐span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length‐dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.     

Ubiquitin‐like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

Ubiquitin and ubiquitin‐like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre‐mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5‐deficient cells display spliceosome‐independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5–FANCI binding, we generated separation‐of‐function mutants to demonstrate that key aspects of FA pathway function, including FANCI–FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5–FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.     

DNA识别受体PYHIN家族及相关病毒免疫逃逸机制的研究

宿主细胞内的DNA识别受体可识别病毒核酸分子并激活细胞天然免疫反应,从而产生抗病毒效应;同时,病毒也进化出相应机制来逃避或抑制这种免疫反应。本文总结了宿主细胞内DNA识别受体PYHIN家族识别病毒核酸并激活细胞天然免疫反应的特点和分子机制,并讨论了病毒逃避宿主天然免疫应答的方式。     

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments

In adenoviral virions, the genome is organized into a chromatin‐like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication‐related factors. We show de novo formation of a subnuclear domain, which we termed Virus‐induced Post‐Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.      

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

The mammalian PYHIN gene family: Phylogeny, evolution and expression

ABSTRACT: BACKGROUND: Proteins of the mammalian PYHIN (IFI200/HIN-200) family are involved in defence against infection through recognition of foreign DNA. The family member absent in melanoma 2 (AIM2) binds cytosolic DNA via its HIN domain and initiates inflammasome formation via its pyrin domain. AIM2 lies within a cluster of related genes, many of which are uncharacterised in mouse. To better understand the evolution, orthology and function of these genes, we have documented the range of PYHIN genes present in representative mammalian species, and undertaken phylogenetic and expression analyses. RESULTS: No PYHIN genes are evident in non-mammals or monotremes, with a single member found in each of three marsupial genomes. Placental mammals show variable family expansions, from one gene in cow to four in human and 14 in mouse. A single HIN domain appears to have evolved in the common ancestor of marsupials and placental mammals, and duplicated to give rise to three distinct forms (HIN-A, -B and -C) in the placental mammal ancestor. Phylogenetic analyses showed that AIM2 HIN-C and pyrin domains clearly diverge from the rest of the family, and it is the only PYHIN protein with orthology across many species. Interestingly, although AIM2 is important in defence against some bacteria and viruses in mice, AIM2 is a pseudogene in cow, sheep, llama, dolphin, dog and elephant. The other 13 mouse genes have arisen by duplication and rearrangement within the lineage, which has allowed some diversification in expression patterns. CONCLUSIONS: The role of AIM2 in forming the inflammasome is relatively well understood, but molecular interactions of other PYHIN proteins involved in defence against foreign DNA remain to be defined. The non-AIM2 PYHIN protein sequences are very distinct from AIM2, suggesting they vary in effector mechanism in response to foreign DNA, and may bind different DNA structures. The PYHIN family has highly varied gene composition between mammalian species due to lineage-specific duplication and loss, which probably indicates different adaptations for fighting infectious disease. Non-genomic DNA can indicate infection, or a mutagenic threat. We hypothesise that defence of the genome against endogenous retroelements has been an additional evolutionary driver for PYHIN proteins.     

Structure–function analysis of ZAR1 immune receptor reveals key molecular interactions for activity

NLR (nucleotide‐binding [NB] leucine‐rich repeat [LRR] receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly modulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor‐like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana that allowed us to demonstrate that ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modelling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions modulate ZAR1 activity. We identified determinants required for the formation of the ZAR^CC oligomer and its activity. Lastly, we characterized intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.