CRISPR Libraries and Whole-Genome Screening to Identify Essential Factors for Viral Infections.

The CRISPR-Cas9 system has revolutionized genetics and offers a simple and inexpensive way of generating perturbation that results in gene repression, activation, or editing. The advances in this technique make possible the development of CRISPR libraries which consist of a set of sgRNAs to cause perturbations in several genes in the same cell population. The use of libraries raised the CRISPR-Cas9 technique to a genomic scale and provides a powerful approach for identifying previously unknown molecular mechanisms and pathways involved in a specific phenotype or biological process. More specifically, the CRISPRko libraries (set of sgRNAs for gene knockout) and their high-throughput screenings are widely used in research with viral agents, and it was enlarged even more with the COVID-19 pandemic. With this chapter, we aim to point out how this tool helps in understanding virus-host relationships, such as the mechanisms of virus entry into the cell, the essential factors for its replication, and the cellular pathways involved in the response against the pathogen. The chapter also provided some practical considerations for each step of an experimentation using these tools that include choosing the library and screening type, the target cell, the viral strain, the library amplification and guaranteeing its coverage, the strategies for the gene screening pipeline by bioinformatics, and finally, target validation. To conclude, it was presented a table reviewing the last updates in the research for antiviral therapies using CRISPR libraries.

Rio Negro Virus Infection, Bolivia, 2021.

In May 2021, an agricultural worker originally from Trementinal, Argentina, sought treatment for febrile illness in Tarija, Bolivia, where he resided at the time of illness onset. The patient tested negative for hantavirus RNA, but next-generation sequencing of a serum sample yielded a complete genome for Rio Negro virus.

Tiny Regulators in Viral Infection: Carving SARS-CoV-2 by miRNAs.

Viruses are microscopic biological entities that can cause diseases. Viruses require a host cell to replicate and generate progeny. Once inside, viruses hijack the main cellular machinery for their benefit, disrupting cell functions and causing detrimental effects on cell physiology. MicroRNAs are short, non-coding RNAs that regulate gene expression. Recent works have shown that cell-miRNAs can modulate antiviral defense during viral infection, and viruses can disrupt these existing miRNA networks. Furthermore, multiple RNA viruses encode their own miRNAs to evade the host immune response. In this review, we analyze the activities of both, miRNAs as pro-viral modulators and miRNAs as anti-viral agents and their relationship with the development of the disease.

Is there a role for HSF1 in viral infections?

Cells undergo numerous processes to adapt to new challenging conditions and stressors. Heat stress is regulated by a family of heat shock factors (HSFs) that initiate a heat shock response by upregulating the expression of heat shock proteins (HSPs) intended to counteract cellular damage elicited by increased environmental temperature. Heat shock factor 1 (HSF1) is known as the master regulator of the heat shock response and upon its activation induces the transcription of genes that encode for molecular chaperones, such as HSP40, HSP70, and HSP90. Importantly, an accumulating body of studies relates HSF1 with viral infections; the induction of fever during viral infection may activate HSF1 and trigger a consequent heat shock response. Here, we review the role of HSF1 in different viral infections and its impact on the health outcome for the host. Studying the relationship between HSF1 and viruses could open new potential therapeutic strategies given the availability of drugs that regulate the activation of this transcription factor.

RNA-Binding Proteins as Regulators of Internal Initiation of Viral mRNA Translation.

Viruses are obligate intracellular parasites that depend on the host's protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. Many viruses utilize non-canonical strategies such as targeting host eIFs and RNA elements known as internal ribosome entry sites (IRESs) to reprogram cellular gene expression, ensuring preferential translation of vRNAs. In this review, we discuss vRNA IRES-mediated translation initiation, highlighting the role of RNA-binding proteins (RBPs), other than the canonical translation initiation factors, in regulating their activity.

An Integrated View of Virus-Triggered Cellular Plasticity Using Boolean Networks.

Virus-related mortality and morbidity are due to cell/tissue damage caused by replicative pressure and resource exhaustion, e.g., HBV or HIV; exaggerated immune responses, e.g., SARS-CoV-2; and cancer, e.g., EBV or HPV. In this context, oncogenic and other types of viruses drive genetic and epigenetic changes that expand the tumorigenic program, including modifications to the ability of cancer cells to migrate. The best-characterized group of changes is collectively known as the epithelial-mesenchymal transition, or EMT. This is a complex phenomenon classically described using biochemistry, cell biology and genetics. However, these methods require enormous, often slow, efforts to identify and validate novel therapeutic targets. Systems biology can complement and accelerate discoveries in this field. One example of such an approach is Boolean networks, which make complex biological problems tractable by modeling data ("nodes") connected by logical operators. Here, we focus on virus-induced cellular plasticity and cell reprogramming in mammals, and how Boolean networks could provide novel insights into the ability of some viruses to trigger uncontrolled cell proliferation and EMT, two key hallmarks of cancer.

Central role of the placenta during viral infection: Immuno-competences and miRNA defensive responses.

Pregnancy is a unique immunological condition in which an "immune-diplomatic" dialogue between trophoblasts and maternal immune cells is established to protect the fetus from rejection, to create a privileged environment in the uterus and to simultaneously be alert to any infectious challenge. The maternal-placental-fetal interface (MPFI) performs an essential role in this immunological defense. In this review, we will address the MPFI as an active immuno-mechanical barrier that protects against viral infections. We will describe the main viral infections affecting the placenta and trophoblasts and present their structure, mechanisms of immunocompetence and defensive responses to viral infections in pregnancy. In particular, we will analyze infection routes in the placenta and trophoblasts and the maternal-fetal outcomes in both. Finally, we will focus on the cellular targets of the antiviral microRNAs from the C19MC cluster, and their effects at both the intra- and extracellular level.

The Aryl Hydrocarbon Receptor as a Modulator of Anti-viral Immunity.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which interacts with a wide range of organic molecules of endogenous and exogenous origin, including environmental pollutants, tryptophan metabolites, and microbial metabolites. The activation of AHR by these agonists drives its translocation into the nucleus where it controls the expression of a large number of target genes that include the AHR repressor (AHRR), detoxifying monooxygenases (CYP1A1 and CYP1B1), and cytokines. Recent advances reveal that AHR signaling modulates aspects of the intrinsic, innate and adaptive immune response to diverse microorganisms. This review will focus on the increasing evidence supporting a role for AHR as a modulator of the host response to viral infection.

Viral metagenomics in blood donations with post-donation illness reports from Brazil.

BACKGROUND: Post-donation illness can be described as appearance of clinical symptoms in blood donors after donation. The consequent call back of the donor to report these symptoms to the blood collection institution is considered a post-donation illness report (PDIR). The most suitable way to examine whether PDIR is related to infection is to apply next-generation sequencing (NGS) and viral metagenomics. Investigation into a PDIR can reveal its importance for transfusion safety and help elaborate strategies for donor education in order to prevent the transfusion transmission of infections which are not routinely tested by the blood collection services. MATERIALS AND METHODS: We applied NGS and viral metagenomics on blood donations which were deferred due to a PDIR. Thirty-three PDIR donations obtained in the Blood Center of Ribeirão Preto, Southeast Brazil, were evaluated. Sequencing was performed using Illumina NextSeq 550 (Illumina Inc, San Diego, CA, USA) equipment and the reads obtained for each sample were analysed by specific bioinformatic pipeline for the classification and discovery of emerging viruses. The identified viral agents by metagenomics were directly confirmed by molecular methods. RESULTS: In all PDIR donations, we found abundant reads of commensal viruses belonging to the Anelloviridae family as well as human pegivirus-1. However, we were also able to identify blood donations positive for clinically important viruses like dengue serotype-2 (DENV-2) of the Asian-American genotype and parvovirus B19 (B19V). Both viruses were also confirmed by real-time polymerase chain reaction, detecting DENV-2 RNA in a significant number of cases (7 samples, 21.2%), compared to B19V which was confirmed in 1 case (3.0%). DISCUSSION: Our study applies for the first time viral metagenomics to evaluate the significance of PDIRs. We confirm the crucial importance of the donor providing a timely PDIR for the prevention of transfusion transmission of viral infections which are not routinely tested in the blood banks worldwide.

siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants.

A non-transgenic approach based on RNA interference was employed to induce protection against tomato mosaic virus (ToMV) infection in tomato plants. dsRNA molecules targeting the cp gene of ToMV were topically applied on plants prior to virus inoculation. Protection was dose-dependent and sequence-specific. While no protection was achieved when 0-16 µg dsRNA were used, maximum rates of resistance (60 and 63%) were observed in doses of 200 and 400 µg/plant, respectively. Similar rates were also obtained against potato virus Y when targeting its cp gene. The protection was quickly activated upon dsRNA application and lasted for up to 4 days. In contrast, no detectable antiviral response was triggered by the dsRNA from a begomovirus genome, suggesting the method is not effective against phloem-limited DNA viruses. Deep sequencing was performed to analyze the biogenesis of siRNA populations. Although long-dsRNA remained in the treated leaves for at least 10 days, its systemic movement was not observed. Conversely, dsRNA-derived siRNA populations (mainly 21- and 22-nt) were detected in non-treated leaves, which indicates endogenous processing and transport through the plant. Altogether, this study provides critical information for the development of novel tools against plant viruses; strengths and limitations inherent to the systems are discussed.

RNA-Seq Data-Mining Allows the Discovery of Two Long Non-Coding RNA Biomarkers of Viral Infection in Humans.

There is a growing interest in unraveling gene expression mechanisms leading to viral host invasion and infection progression. Current findings reveal that long non-coding RNAs (lncRNAs) are implicated in the regulation of the immune system by influencing gene expression through a wide range of mechanisms. By mining whole-transcriptome shotgun sequencing (RNA-seq) data using machine learning approaches, we detected two lncRNAs (ENSG00000254680 and ENSG00000273149) that are downregulated in a wide range of viral infections and different cell types, including blood monocluclear cells, umbilical vein endothelial cells, and dermal fibroblasts. The efficiency of these two lncRNAs was positively validated in different viral phenotypic scenarios. These two lncRNAs showed a strong downregulation in virus-infected patients when compared to healthy control transcriptomes, indicating that these biomarkers are promising targets for infection diagnosis. To the best of our knowledge, this is the very first study using host lncRNAs biomarkers for the diagnosis of human viral infections.

Strategies for Success. Viral Infections and Membraneless Organelles.

Regulation of RNA homeostasis or "RNAstasis" is a central step in eukaryotic gene expression. From transcription to decay, cellular messenger RNAs (mRNAs) associate with specific proteins in order to regulate their entire cycle, including mRNA localization, translation and degradation, among others. The best characterized of such RNA-protein complexes, today named membraneless organelles, are Stress Granules (SGs) and Processing Bodies (PBs) which are involved in RNA storage and RNA decay/storage, respectively. Given that SGs and PBs are generally associated with repression of gene expression, viruses have evolved different mechanisms to counteract their assembly or to use them in their favor to successfully replicate within the host environment. In this review we summarize the current knowledge about the viral regulation of SGs and PBs, which could be a potential novel target for the development of broad-spectrum antiviral therapies.

Eukaryotic initiation factor 4A (eIF4A) during viral infections.

The helicase eIF4A is part of the cellular eIF4F translation initiation complex. The main functions of eIF4A are to remove secondary complex structures within the 5'-untranslated region and to displace proteins attached to mRNA. As intracellular parasites, viruses regulate the processes involved in protein synthesis, and different mechanisms related to controlling translation factors, such as eIF4A, have been found. The inhibitors of this factor are currently known; these substances could be used in the near future as part of antiviral pharmacological therapies in instances of replication cycles in which eIF4A is required. In this review, the particularities of how some viruses make use of this initiation factor to synthesize their proteins are discussed.

DNA Related Enzymes as Molecular Targets for Antiviral and Antitumoral Chemotherapy. A Natural Overview of the Current Perspectives.

BACKGROUND: The discovery of new chemotherapeutic agents still remains a continuous goal to achieve. DNA polymerases and topoisomerases act in nucleic acids metabolism modulating different processes like replication, mitosis, damage repair, DNA topology and transcription. It has been widely documented that Polymerases serve as molecular targets for antiviral and antitumoral chemotherapy. Furthermore, telomerase is a ribonucleoprotein with exacerbated activity in most of the tumor cell lines, becoming as an emergent target in Cancer treatment. METHODS: We undertook an exhaustive search of bibliographic databases for peer-reviewed research literature related to the last decade. The characteristics of screened bibliography describe structure activity relationships and show the principal moieties involved. This work tries to summarize the investigation about natural and semi-synthetic products with natural origin with the faculty to inhibit key enzymes that play a crucial role in DNA metabolism. RESULTS: Eighty-five data references were included in this review, showing natural products widely distributed throughout the plant kingdom and their bioactive properties such as tumor growing inhibitory effects, and anti-AIDS activity. CONCLUSION: The findings of this review confirm the importance to find new drugs and biologically active natural products, and their potential medicinally useful benefits.

The role of myeloid-derived suppressor cells in chronic infectious diseases and the current methodology available for their study.

An effective pathogen has the ability to evade the immune response. The strategies used to achieve this may be based on the direct action of virulence factors or on the induction of host factors. Myeloid-derived suppressor cells (MDSCs) are immune cells with an incredible ability to suppress the inflammatory response, which makes them excellent targets to be exploited by pathogenic bacteria, viruses, or parasites. In this review, we describe the origin and suppressive mechanisms of MDSCs, as well as their role in chronic bacterial, viral, and parasitic infections, where their expansion seems to be essential in the chronicity of the disease. We also analyze the disadvantages of current MDSC depletion strategies and the different in vitro generation methods, which can be useful tools for the deeper study of these cells in the context of microbial infections.

NOD2: Activation During Bacterial and Viral Infections, Polymorphisms and Potential as Therapeutic Target.

Nucleotide-binding domain (NBD) leucine-rich repeat (LRR)-containing receptors or NLRs are a family of receptors that detect both, molecules associated to pathogens and alarmins, and are located mainly in the cytoplasm. NOD2 belongs to the NLR family and is a dynamic receptor capable of interacting with multiple proteins and modulate immune responses in a stimuli-dependent manner. The experimental evidence shows that interaction between NOD2 structural domains and the effector proteins shape the overall response against bacterial or viral infections. Other reports have focused on the importance of NOD2 not only in infection but also in maintaining tissue homeostasis. However, not only protein interactions relate to function but also certain polymorphisms in the gene that encodes NOD2 have been associated with inflammatory diseases, such as Crohn's disease. Here, we review the importance and general characteristics of NOD2, discussing its participation in infections caused by bacteria and viruses as well as its interaction with other pathogen recognition receptors or effectors to induce antibacterial and antiviral responses. Finally, the role of NOD2 in chronic inflammatory conditions and its potential to be targeted therapeutically are examined.

CDK9: A key player in cancer and other diseases.

Cyclin-Dependent Kinase 9 (CDK9) is part of a functional diverse group of enzymes responsible for cell cycle control and progression. It associates mainly with Cyclin T1 and forms the Positive Transcription Elongation Factor b (p-TEFb) complex responsible for regulation of transcription elongation and mRNA maturation. Recent studies have highlighted the importance of CDK9 in many relevant pathologic processes, like cancer, cardiovascular diseases, and viral replication. Herein we provide an overview of the different pathways in which CDK9 is directly and indirectly involved.

Beyond ISGlylation: Functions of Free Intracellular and Extracellular ISG15.

ISG15 is a ubiquitin-like type I IFN-stimulated protein of 15 kDa and is one of the most prominently expressed proteins in viral infections. ISG15 is widely known to be involved in a process called ISGylation, where it binds to over 150 targets from a variety of classes of proteins including central immune signaling pathways such as those mediated by NFκB, JNK, and IRF-3. However, ISG15 also exists in a free form that can act intra- or extracellularly. In vitro and in vivo evidences suggest that free ISG15 play different roles in several cellular processes, from cancer and defense against viral infections to activation of immune cells such as lymphocytes, monocytes, and NK cells. This review discusses the roles of free intracellular and secreted ISG15 approaching questions yet to be answered about the mechanism of action of this protein.