A Guideline Strategy for Identifying Genes/Proteins Regulating Antiviral Innate Immunity.

Antiviral innate immunity is a complicated system initiated by the induction of type I interferon (IFN-I) and downstream interferon-stimulated genes (ISGs) and is finely regulated by numerous positive and negative factors at different signaling adaptors. During this process, posttranslational modifications, especially ubiquitination, are the most common regulatory strategy used by the host to switch the antiviral innate signaling pathway and are mainly controlled by E3 ubiquitin ligases from different protein families. A comprehensive understanding of the regulatory mechanisms and a novel discovery of regulatory factors involved in the IFN-I signaling pathway are important for researchers to identify novel therapeutic targets against viral infectious diseases based on innate immunotherapy. In this section, we use the E3 ubiquitin ligase as an example to guide the identification of a protein belonging to the RING Finger (RNF) family that regulates the RIG-I-mediated IFN-I pathway through ubiquitination.

CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.

The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.

CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

A Guideline Strategy for Identifying a Viral Gene/Protein Evading Antiviral Innate Immunity.

Antiviral innate immunity is the first line of defence against viruses. The interferon (IFN) signaling pathway, the DNA damage response (DDR), apoptosis, endoplasmic reticulum (ER) stress, and autophagy are involved in antiviral innate immunity. Viruses abrogate the antiviral immune response of cells to replication in various ways. Viral genes/proteins play a key role in evading antiviral innate immunity. Here, we will discuss the interference of viruses with antiviral innate immunity and the strategy for identifying viral gene/protein immune evasion.

Transcriptomics in the Study of Antiviral Innate Immunity.

Transcriptomics is an extremely important area of molecular biology and is a powerful tool for studying all RNA molecules in an organism. Conventional transcriptomic technologies include microarrays and RNA sequencing, and the rapid development of single-cell sequencing and spatial transcriptomics in recent years has provided an enormous scope for research in this field. This chapter describes the application, significance, and experimental procedures of a variety of transcriptomic technologies in antiviral natural immunity.

Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

DNA PAMPs as Molecular Tools for the cGAS-STING Signaling Pathways.

Beyond its role as the bearer of genetic material, DNA also plays a crucial role in the activation phase of innate immunity. Pathogen recognition receptors (PRRs) and their homologs, pathogen-associated molecular patterns (PAMPs), form the foundation for driving innate immune activation and the induction of immune responses during infection. In the context of DNA viruses or bacterial infections, specific DNA sequences are recognized and bound by DNA sensors, marking the DNA as a PAMP for host recognition and subsequent activation of innate immunity. The primary DNA sensor pathway known to date is cGAS-STING, which can induce Type I interferons (IFN) and innate immune responses against viruses and bacteria. Additionally, the cGAS-STING pathway has been identified to mediate functions in autophagy and senescence. Herein, we introduce methods for using DNA PAMPs as molecular tools to study the role of cGAS-STING and its signaling pathway in regulating innate immunity, both in vitro and in vivo.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

Identifying RNA Sensors in Antiviral Innate Immunity.

The innate immune system plays a pivotal role in pathogen recognition and the initiation of innate immune responses through its Pathogen Recognition Receptors (PRRs), which detect Pathogen-Associated Molecular Patterns (PAMPs). Nucleic acids, including RNA and DNA, are recognized as particularly significant PAMPs, especially in the context of viral pathogens. During RNA virus infections, specific sequences in the viral RNA mark it as non-self, enabling host recognition through interactions with RNA sensors, thereby triggering innate immunity. Given that some of the most lethal viruses are RNA viruses, they pose a severe threat to human and animal health. Therefore, understanding the immunobiology of RNA PRRs is crucial for controlling pathogen infections, particularly RNA virus infections. In this chapter, we will introduce a "pull-down" method for identifying RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensors.

Cryo-Electron Microscopy in the Study of Antiviral Innate Immunity.

Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.

Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.

The Application of Organoids in the Study of Antiviral Innate Immunity.

Antiviral innate immunity plays a critical role in the defense against viral infections, yet its complex interactions with viruses have been challenging to study using traditional models. Organoids, three-dimensional (3D) tissue-like structures derived from stem cells, have emerged as powerful tools for modeling human tissues and studying the complex interactions between viruses and the host innate immune system. This chapter summarizes relevant applications of organoids in antiviral innate immunity studies and provides detailed information and experimental procedures for using organoids to study antiviral innate immunity.

Epidemiological Study in Antiviral Innate Immunity.

This chapter summarizes the epidemiological study design of natural immune epidemiology studies based on recent COVID-19-related research. The epidemiological studies on antiviral innate immunity have mainly included randomized controlled trials (RCTs) and observational studies. Importantly, this chapter will discuss how to use these methodologies to answer an epidemiological question of natural immunity in the viral infection process based on previous studies. An observational case- or cohort-based study of antiviral innate immunity may support this theoretical hypothesis but is not appropriate for clinical practice or treatment. RCTs are the gold standard for epidemiological studies and occupy a greater role in the hierarchy of evidence.

Yeast Two-Hybrid Assay for Investigating Antiviral Innate Immunity.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

Identifying a Gene Deficiency in the Antiviral Innate Immune Signaling Pathway.

Innate immunity is an important defense barrier for the human body. After viral pathogen-associated molecular patterns (PAMPs) are detected by host-pathogen recognition receptors (PRRs), the associated signaling pathways trigger the activation of the interferon (IFN) regulatory factor (IRF) family members and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). However, any gene defects among the signaling adaptors will compromise innate immune efficiency. Therefore, investigating genetic defects in the antiviral innate immune signaling pathway is important. We summarize the commonly used research methods related to antiviral immune gene defects and outline the relevant research protocols, which will help investigators study antiviral innate immunity.

Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

The battle between host antiviral innate immunity and immune evasion by cytomegalovirus.

Cytomegalovirus (CMV) has successfully established a long-lasting latent infection in humans due to its ability to counteract the host antiviral innate immune response. During coevolution with the host, the virus has evolved various evasion techniques to evade the host's innate immune surveillance. At present, there is still no vaccine available for the prevention and treatment of CMV infection, and the interaction between CMV infection and host antiviral innate immunity is still not well understood. However, ongoing studies will offer new insights into how to treat and prevent CMV infection and its related diseases. Here, we update recent studies on how CMV evades antiviral innate immunity, with a focus on how CMV proteins target and disrupt critical adaptors of antiviral innate immune signaling pathways. This review also discusses some classic intrinsic cellular defences that are crucial to the fight against viral invasion. A comprehensive review of the evasion mechanisms of antiviral innate immunity by CMV will help investigators identify new therapeutic targets and develop vaccines against CMV infection.

The crosstalk between DNA-damage responses and innate immunity.

DNA damage is typically caused during cell growth by DNA replication stress or exposure to endogenous or external toxins. The accumulation of damaged DNA causes genomic instability, which is the root cause of many serious disorders. Multiple cellular organisms utilize sophisticated signaling pathways against DNA damage, collectively known as DNA damage response (DDR) networks. Innate immune responses are activated following cellular abnormalities, including DNA damage. Interestingly, recent studies have indicated that there is an intimate relationship between the DDR network and innate immune responses. Diverse kinds of cytosolic DNA sensors, such as cGAS and STING, recognize damaged DNA and induce signals related to innate immune responses, which link defective DDR to innate immunity. Moreover, DDR components operate in immune signaling pathways to induce IFNs and/or a cascade of inflammatory cytokines via direct interactions with innate immune modulators. Consistently, defective DDR factors exacerbate the innate immune imbalance, resulting in severe diseases, including autoimmune disorders and tumorigenesis. Here, the latest progress in understanding crosstalk between the DDR network and innate immune responses is reviewed. Notably, the dual function of innate immune modulators in the DDR network may provide novel insights into understanding and developing targeted immunotherapies for DNA damage-related diseases, even carcinomas.