Beyond pathogens: the intriguing genetic legacy of endogenous retroviruses in host physiology.

The notion that viruses played a crucial role in the evolution of life is not a new concept. However, more recent insights suggest that this perception might be even more expansive, highlighting the ongoing impact of viruses on host evolution. Endogenous retroviruses (ERVs) are considered genomic remnants of ancient viral infections acquired throughout vertebrate evolution. Their exogenous counterparts once infected the host's germline cells, eventually leading to the permanent endogenization of their respective proviruses. The success of ERV colonization is evident so that it constitutes 8% of the human genome. Emerging genomic studies indicate that endogenous retroviruses are not merely remnants of past infections but rather play a corollary role, despite not fully understood, in host genetic regulation. This review presents some evidence supporting the crucial role of endogenous retroviruses in regulating host genetics. We explore the involvement of human ERVs (HERVs) in key physiological processes, from their precise and orchestrated activities during cellular differentiation and pluripotency to their contributions to aging and cellular senescence. Additionally, we discuss the costs associated with hosting a substantial amount of preserved viral genetic material.

Single-cell RNA-seq revealed heterogeneous responses and functional differentiation of hemocytes against white spot syndrome virus infection in Litopenaeus vannamei.

Shrimp hemocytes are the vital immune cells participating in innate immune response to defend against viruses. However, the lack of specific molecular markers for shrimp hemocyte hindered the insightful understanding of their functional clusters and differential roles in combating microbial infections. In this study, we used single-cell RNA sequencing to map the transcriptomic landscape of hemocytes from the white spot syndrome virus (WSSV)-infected Litopenaeus vannamei and conjointly analyzed with our previous published single-cell RNA sequencing technology data from the healthy hemocytes. A total of 16 transcriptionally distinct cell clusters were identified, which occupied different proportions in healthy and WSSV-infected hemocytes and exerted differential roles in antiviral immune response. Following mapping of the sequencing data to the WSSV genome, we found that all types of hemocytes could be invaded by WSSV virions, especially the cluster 8, which showed the highest transcriptional levels of WSSV genes and exhibited a cell type-specific antiviral response to the viral infection. Further evaluation of the cell clusters revealed the delicate dynamic balance between hemocyte immune response and viral infestation. Unsupervised pseudo-time analysis of hemocytes showed that the hemocytes in immune-resting state could be significantly activated upon WSSV infection and then functionally differentiated to different hemocyte subsets. Collectively, our results revealed the differential responses of shrimp hemocytes and the process of immune-functional differentiation post-WSSV infection, providing essential resource for the systematic insight into the synergistic immune response mechanism against viral infection among hemocyte subtypes. IMPORTANCE: Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.

Mucosal host-microbe interactions associate with clinical phenotypes in inflammatory bowel disease.

Disrupted host-microbe interactions at the mucosal level are key to the pathophysiology of IBD. This study aimed to comprehensively examine crosstalk between mucosal gene expression and microbiota in patients with IBD. To study tissue-specific interactions, we perform transcriptomic (RNA-seq) and microbial (16S-rRNA-seq) profiling of 697 intestinal biopsies (645 derived from 335 patients with IBD and 52 from 16 non-IBD controls). Mucosal gene expression patterns in IBD are mainly determined by tissue location and inflammation, whereas the mucosal microbiota composition shows a high degree of individual specificity. Analysis of transcript-bacteria interactions identifies six distinct groups of inflammation-related pathways that are associated with intestinal microbiota (adjusted P < 0.05). An increased abundance of Bifidobacterium is associated with higher expression of genes involved in fatty acid metabolism, while Bacteroides correlates with increased metallothionein signaling. In patients with fibrostenosis, a transcriptional network dominated by immunoregulatory genes is associated with Lachnoclostridium bacteria in non-stenotic tissue (adjusted P < 0.05), while being absent in CD without fibrostenosis. In patients using TNF-α-antagonists, a transcriptional network dominated by fatty acid metabolism genes is linked to Ruminococcaceae (adjusted P < 0.05). Mucosal microbiota composition correlates with enrichment of intestinal epithelial cells, macrophages, and NK-cells. Overall, these data demonstrate the presence of context-specific mucosal host-microbe interactions in IBD, revealing significantly altered inflammation-associated gene-taxa modules, particularly in patients with fibrostenotic CD and patients using TNF-α-antagonists. This study provides compelling insights into host-microbe interactions that may guide microbiota-directed precision medicine and fuels the rationale for microbiota-targeted therapeutics as a strategy to alter disease course in IBD.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

MicroRNA-22-3p displaces critical host factors from the 5' UTR and inhibits the translation of Coxsackievirus B3 RNA.

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Single-cell RNA sequencing to understand host-virus interactions.

Single-cell RNA sequencing (scRNA-seq) has allowed for the profiling of host and virus transcripts and host-virus interactions at single-cell resolution. This review summarizes the existing scRNA-seq technologies together with their strengths and weaknesses. The applications of scRNA-seq in various virological studies are discussed in depth, which broaden the understanding of the immune atlas, host-virus interactions, and immune repertoire. scRNA-seq can be widely used for virology in the near future to better understand the pathogenic mechanisms and discover more effective therapeutic strategies.

Viral RNA Is a Hub for Critical Host-Virus Interactions.

RNA is a central molecule in the life cycle of viruses, acting not only as messenger (m)RNA but also as a genome. Given these critical roles, it is not surprising that viral RNA is a hub for host-virus interactions. However, the interactome of viral RNAs remains largely unknown. This chapter discusses the importance of cellular RNA-binding proteins in virus infection and the emergent approaches developed to uncover and characterise them.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB.

IMPORTANCE: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

IMPORTANCE: The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression.

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.

SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells.

Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.

ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

EBV Reactivation from Latency Is a Degrading Experience for the Host.

During reactivation from latency, gammaherpesviruses radically restructure their host cell to produce virion particles. To achieve this and thwart cellular defenses, they induce rapid degradation of cytoplasmic mRNAs, suppressing host gene expression. In this article, we review mechanisms of shutoff by Epstein-Barr virus (EBV) and other gammaherpesviruses. In EBV, canonical host shutoff is accomplished through the action of the versatile BGLF5 nuclease expressed during lytic reactivation. We explore how BGLF5 induces mRNA degradation, the mechanisms by which specificity is achieved, and the consequences for host gene expression. We also consider non-canonical mechanisms of EBV-induced host shutoff. Finally, we summarize the limitations and barriers to accurate measurements of the EBV host shutoff phenomenon.

SARS-CoV-2 Mutations Lead to a Decrease in the Number of Tissue-Specific MicroRNA-Binding Regions in the Lung.

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides.

Single Particle Analysis of H3N2 Influenza Entry Differentiates the Impact of the Sialic Acids (Neu5Ac and Neu5Gc) on Virus Binding and Membrane Fusion.

Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Two-Color CRISPR Imaging Reveals Dynamics of Herpes Simplex Virus 1 Replication Compartments and Virus-Host Interactions.

Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.