Neuroinflammation During RNA Viral Infections.

Neurotropic RNA viruses continue to emerge and are increasingly linked to diseases of the central nervous system (CNS) despite viral clearance. Indeed, the overall mortality of viral encephalitis in immunocompetent individuals is low, suggesting efficient mechanisms of virologic control within the CNS. Both immune and neural cells participate in this process, which requires extensive innate immune signaling between resident and infiltrating cells, including microglia and monocytes, that regulate the effector functions of antiviral T and B cells as they gain access to CNS compartments. While these interactions promote viral clearance via mainly neuroprotective mechanisms, they may also promote neuropathology and, in some cases, induce persistent alterations in CNS physiology and function that manifest as neurologic and psychiatric diseases. This review discusses mechanisms of RNA virus clearance and neurotoxicity during viral encephalitis with a focus on the cytokines essential for immune and neural cell inflammatory responses and interactions. Understanding neuroimmune communications in the setting of viral infections is essential for the development of treatments that augment neuroprotective processes while limiting ongoing immunopathological processes that cause ongoing CNS disease.

Sixty Years of Discovery.

Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

Translation and Replication Dynamics of Single RNA Viruses.

RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.

Exploring the expanding universe of host-virus interactions mediated by viral RNA.

RNA is a central molecule in RNA virus biology; however, the interactions that it establishes with the host cell are only starting to be elucidated. In recent years, a methodology revolution has dramatically expanded the scope of host-virus interactions involving the viral RNA (vRNA). A second wave of method development has enabled the precise study of these protein-vRNA interactions in a life cycle stage-dependent manner, as well as providing insights into the interactome of specific vRNA species. This review discusses these technical advances and describes the new regulatory mechanisms that have been identified through their use. Among these, we discuss the importance of vRNA in regulating protein function through a process known as riboregulation. We envision that the elucidation of vRNA interactomes will open new avenues of research, including pathways to the discovery of host factors with therapeutic potential against viruses.

DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.

Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism.

The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

A novel DCL2-dependent miRNA pathway in tomato affects susceptibility to RNA viruses.

Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.

Covert RNA viruses in medflies differ in their mode of transmission and tissue tropism.

Numerous studies have demonstrated the presence of covert viral infections in insects. These infections can be transmitted in insect populations via two main routes: vertical from parents to offspring, or horizontal between nonrelated individuals. Thirteen covert RNA viruses have been described in the Mediterranean fruit fly (medfly). Some of these viruses are established in different laboratory-reared and wild medfly populations, although variations in the viral repertoire and viral levels have been observed at different time points. To better understand these viral dynamics, we characterized the prevalence and levels of covert RNA viruses in two medfly strains, assessed the route of transmission of these viruses, and explored their distribution in medfly adult tissues. Altogether, our results indicated that the different RNA viruses found in medflies vary in their preferred route of transmission. Two iflaviruses and a narnavirus are predominantly transmitted through vertical transmission via the female, while a nodavirus and a nora virus exhibited a preference for horizontal transmission. Overall, our results give valuable insights into the viral tropism and transmission of RNA viruses in the medfly, contributing to the understanding of viral dynamics in insect populations. IMPORTANCE: The presence of RNA viruses in insects has been extensively covered. However, the study of host-virus interaction has focused on viruses that cause detrimental effects to the host. In this manuscript, we uncovered which tissues are infected with covert RNA viruses in the agricultural pest Ceratitis capitata, and which is the preferred transmission route of these viruses. Our results showed that vertical and horizontal transmission can occur simultaneously, although each virus is transmitted more efficiently following one of these routes. Additionally, our results indicated an association between the tropism of the RNA virus and the preferred route of transmission. Overall, these results set the basis for understanding how viruses are established and maintained in medfly populations.

Zinc-finger CCHC-type containing protein 8 promotes RNA virus replication by suppressing the type-I interferon responses.

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective.IMPORTANCEThe innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses.

RIG-I detects viral genomic RNA during negative-strand RNA virus infection.

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

Varroa destructor parasitism and Deformed wing virus infection in honey bees are linked to peroxisome-induced pathways.

The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-ß signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.

The regulation of cGAS-STING signaling by RNA virus-derived components.

The Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) serves as a key innate immune signaling axis involved in the regulation of various human diseases. It has been found that cGAS-STING pathway can recognize a variety of cytosolic double-stranded DNA (dsDNA), contributing to cause a robust type I interferon response thereby affecting the occurrence and progression of viral infection. Accumulating evidence indicates RNA virus-derived components play an important role in regulating cGAS-STING signaling, either as protective or pathogenic factors in the pathogenesis of diseases. Thus, a comprehensive understanding of the function of RNA virus-derived components in regulating cGAS-STING signaling will provide insights into developing novel therapies. Here, we review the existing literature on cGAS-STING pathway regulated by RNA virus-derived components to propose insights into pharmacologic strategies targeting the cGAS-STING pathway.

Two +ssRNA mycoviruses cohabiting the fungal cultivar of leafcutter ants.

Leafcutter ants are dominant herbivores in the Neotropics and rely on a fungus (Leucoagaricus gongylophorus) to transform freshly gathered leaves into a source of nourishment rather than consuming the vegetation directly. Here we report two virus-like particles that were isolated from L. gongylophorus and observed using transmission electron microscopy. RNA sequencing identified two +ssRNA mycovirus strains, Leucoagaricus gongylophorus tymo-like virus 1 (LgTlV1) and Leucoagaricus gongylophorus magoulivirus 1 (LgMV1). Genome annotation of LgTlV1 (7401 nt) showed conserved domains for methyltransferase, endopeptidase, viral RNA helicase, and RNA-dependent RNA polymerase (RdRp). The smaller genome of LgMV1 (2636 nt) contains one open reading frame encoding an RdRp. While we hypothesize these mycoviruses function as symbionts in leafcutter farming systems, further study will be needed to test whether they are mutualists, commensals, or parasites.

Learning performance and GABAergic pathway link to deformed wing virus in the mushroom bodies of naturally infected honey bees.

Viral infections can be detrimental to the foraging ability of the western honey bee, Apis mellifera. The deformed wing virus (DWV) is the most common honey bee virus and has been proposed as a possible cause of learning and memory impairment. However, evidence for this phenomenon so far has come from artificially infected bees, while less is known about the implications of natural infections with the virus. Using the proboscis extension reflex (PER), we uncovered no significant association between a simple associative learning task and natural DWV load. However, when assessed through a reversal associative learning assay, bees with higher DWV load performed better in the reversal learning phase. DWV is able to replicate in the honey bee mushroom bodies, where the GABAergic signalling pathway has an antagonistic effect on associative learning but is crucial for reversal learning. Hence, we assessed the pattern of expression of several GABA-related genes in bees with different learning responses. Intriguingly, mushroom body expression of selected genes was positively correlated with DWV load, but only for bees with good reversal learning performance. We hypothesise that DWV might improve olfactory learning performance by enhancing the GABAergic inhibition of responses to unrewarded stimuli, which is consistent with the behavioural patterns that we observed. However, at higher disease burdens, which might be induced by an artificial infection or by a severe, natural Varroa infestation, this DWV-associated increase in GABA signalling could impair associative learning as previously reported by other studies.

Caught in the act: the invasion of a viral vector changes viral prevalence and titre in native honeybees and bumblebees.

Novel transmission routes change pathogen landscapes and may facilitate disease emergence. The varroa mite is a virus vector that switched to western honeybees at the beginning of the last century, leading to hive mortality, particularly in combination with RNA viruses. A recent invasion of varroa on the French island of Ushant introduced vector-mediated transmission to one of the last varroa-naive native honeybee populations and caused rapid changes in the honeybee viral community. These changes were characterized by a drastic increase in deformed wing virus type B prevalence and titre in honeybees, as well as knock-on effects in bumblebees, particularly in the year following the invasion. Slow bee paralysis virus also appeared in honeybees and bumblebees, with a 1 year delay, while black queen cell virus declined in honeybees. This study highlights the rapid and far-reaching effects of vector-borne transmission that can extend beyond the directly affected host species, and that the direction of the effect depends on the pathogen's virulence.

Solenopsis richteri (Hymenoptera: Formicidae) alates infected with deformed wing virus display wing deformity with altered mobility.

Deformed wing virus (DWV) has long been identified as a critical pathogen affecting honeybees, contributing to colony losses through wing deformities, neurological impairments, and reduced lifespan. Since DWV also affects other pollinators, it poses a significant threat to global pollination networks. While honeybees have been the focal point of DWV studies, emerging research indicates that this RNA virus is not host-specific but rather a generalist pathogen capable of infecting a wide range of insect species, including other bee species such as bumblebees and solitary bees, as well as wasps and ants. This expands the potential impact of DWV beyond honeybees to broader ecological communities. The black imported fire ant, Solenopsis richteri, is an economically important invasive ant species. In this study, we describe deformed wing (DW) symptoms in S. richteri. DW alates were found in three of nine (33%) laboratory colonies. The symptoms ranged from severely twisted wings to a single crumpled wing tip. Additionally, numerous symptomatic alates also displayed altered mobility, ranging from an ataxic gait to an inability to walk. Viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. richteri can be an alternative host for DWV, expanding our understanding of DWV as a generalist pathogen in insects. However, additional research is required to determine whether DWV is the etiological agent responsible for DW syndrome in S. richteri.

Effect of Solenopsis invicta virus 3 on brood mortality and egg hatch in Solenopsis invicta.

Solenopsis invicta virus 3 (SINV-3) has been shown to cause significant mortality among all stages of its host, Solenopsis invicta. One impact of the virus is alteration of worker ant foraging behavior, which results in colony starvation and collapse over time. Additionally, it has been hypothesized that SINV-3 infection of S. invicta may disrupt worker ant brood care behavior. To investigate this possibility, various combinations of SINV-3-infected and -uninfected adult (worker) and immature (brood) stages were placed together and monitored using the response variables, mortality, egg hatch, and virus load. While significant differences in percent cumulative S. invicta worker ant mortality among six combinations of SINV-3-infected and -uninfected stages were observed, no significant differences in percent cumulative mortality of S. invicta larvae or pupae were observed. No significant differences in egg hatch were observed among SINV-3-uninfected, SINV-3-infected (colony-treated and queen-treated), and starved colonies. Eggs hatched normally in 10-12 days for all treatments indicating that egg care by worker ants was unaffected by SINV-3 infection status. The study further clarifies SINV-3 pathogenesis in its host, S. invicta. Larval mortality in SINV-3-infected colonies does not appear to be caused by worker ant neglect. S. invicta brood under the care of SINV-3-infected worker ants did not exhibit higher mortality rates compared with those tended by SINV-3-uninfected worker ants.