Biology subjects

Johnson, K. N.

Publications and source records attributed to Johnson, K. N..

7 recordsLinked to original sources

bioRxiv2025

Wolbachia-mediated antiviral protection is driven by its multimodal effects on Drosophila melanogaster metabolism

Wolbachia, a common endosymbiont of Drosophila melanogaster, provides a profound antiviral effect in insects. Wolbachia-mediated viral interference has been employed to limit the spread of arboviruses, including dengue. However, the mechanisms underpinning Wolbachia-mediated viral interference are not consistently understood. Previous studies have identified resource competition as a potential mechanism by which Wolbachia disrupts viral replication. Our study uses nuclear magnetic resonance (NMR)-based metabolomics to characterize the bi- and tripartite host-Wolbachia-virus interactions using the model insect Drosophila melanogaster, the protective Wolbachia strain wMel and the pathogenic Drosophila C virus (DCV). The findings reveal that wMel-infected flies showed increased simple carbohydrate catabolism relative to uninfected Drosophila. DCV infection perturbed nucleotide synthesis and nucleotide abundance in Drosophila compared to uninfected Drosophila, driving metabolism to likely meet the viral replication demands imposed on the host. Notably, co-infected Drosophila exhibited a metabolic profile more similar to wMel-infected flies than DCV-infected flies, suggesting that wMel drives metabolism in a direction that at least temporarily inhibits DCV replication. The metabolic profile is indicative of a hypoxic environment that has been known to trigger immune pathways that further contribute to Wolbachia-mediated pathogen blocking. It is probable that Wolbachia-mediated antiviral protection is a multimodal consequence of wMels influence on host metabolism, rather than a single mechanism. These findings will guide future research and contribute to the continued success of Wolbachia-based vector control strategies against RNA arboviruses, potentially leading to novel approaches for defending against such pathogens and improving vector control strategies. Significance StatementWolbachia-mediated antiviral protection has been used extensively to control the spread of insect-borne viruses, with the release of Wolbachia-infected mosquitoes eradicating local dengue transmission in some regions. Despite this real-world impact, how Wolbachia disrupted viruses remained unclear, with three common mechanisms predicted: (1) competition between Wolbachia and virus for host metabolites, or stimulation of host antiviral pathways via (2) immune priming and/or (3) the RNA interference pathway. Our findings reveal that in Drosophila melanogaster, Wolbachia does compete for some metabolites, but it exerts a greater effect by altering host metabolism which in turn creates an intracellular environment unfavorable for viral replication and indirectly triggers an immune response. Thus, the mechanism by which Wolbachia disrupts viruses relies on both metabolic and immune pathways. Understanding these metabolic mechanisms is crucial for optimizing and sustaining this vector control strategy and for shaping future efforts to combat vector-borne diseases.

systems biology↗

bioRxiv2024

Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured ("naive") rats. Six-hour treatment with the specific Gs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator, forskolin, induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12-24 hours later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to -45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from -69 to -66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and indications of reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, and protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. An interesting possibility is that these mechanisms can also be reactivated by re-exposure to inflammatory mediators such as serotonin during subsequent challenges to bodily integrity, "reconsolidating" the cellular memory and thereby extending the duration of persistent nociceptor hyperexcitability. HighlightsO_LINociceptor long-term hyperexcitability (LTH) can be induced by a 5-HT4R agonist. C_LIO_LI5-HT4R-induced LTH manifests as ongoing activity during modest depolarization. C_LIO_LIEnhanced ongoing activity is associated with long-term potentiation of DSFs. C_LIO_LIInduction of LTH depends upon PKA, CREB, transcription, and protein synthesis. C_LIO_LINociceptor LTH may be triggered by conserved memory-related plasticity mechanisms. C_LI

neuroscience↗

bioRxiv2024

Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase. Author SummaryHenipaviruses, such as the prototypical Nipah virus and Hendra virus, are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, as well as for the emergent species, Ghana virus. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, application of this system to Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and identify two orally-efficacious antivirals effective against it. Further, we compare the compatibility of divergent henipavirus replicases with heterotypic viral genetic elements, providing valuable insights for how these species have evolved. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.

microbiology↗

bioRxiv2024

Effects of spontaneous mutations on survival and reproduction of Drosophila serrata infected with Drosophila C virus

The impact of selection on host immune function genes has been widely documented. However, it remains essentially unknown how mutation influences the quantitative immune traits that selection acts on. Applying a classical mutation accumulation (MA) experimental design in Drosophila serrata, we found the mutational variation in susceptibility (median time of death, LT50) to Drosophila C virus (DCV) was of similar magnitude to that reported for intrinsic survival traits. However, the mean LT50 did not change as mutations accumulated, suggestion no directional bias in mutational effects. Maintenance of genetic variance in immune function is hypothesised to be influenced by pleiotropic effects on immunity and other traits that contribute to fitness. To investigate this, we assayed female reproductive output for a subset of MA lines with relatively long or short survival times under DCV infection. Longer survival time tended to be associated with lower reproductive output, suggesting that mutations affecting susceptibility to DCV had pleiotropic effects on investment in reproductive fitness. Further studies are needed to uncover the general patterns of mutational effect on immune responses and other fitness traits, and to determine how selection might typically act on new mutations via their direct and pleiotropic effects.

evolutionary biology↗

bioRxiv2024

Widespread latent hyperactivity of nociceptors outlasts enhanced avoidance behavior following incision injury

Nociceptors with somata in dorsal root ganglia (DRGs) exhibit an unusual readiness to switch from an electrically silent state to a hyperactive state of tonic, nonaccommodating, low-frequency, irregular discharge of action potentials (APs). Ongoing activity (OA) during this state is present in vivo in rats months after spinal cord injury (SCI), and has been causally linked to SCI pain. OA induced by various neuropathic conditions in rats, mice, and humans is retained in nociceptor somata after dissociation and culturing, providing a powerful tool for investigating its mechanisms and functions. An important question is whether similar nociceptor OA is induced by painful conditions other than neuropathy. The present study shows that probable nociceptors dissociated from DRGs of rats subjected to postsurgical pain (induced by plantar incision) exhibit OA. The OA was most apparent when the soma was artificially depolarized to a level within the normal range of membrane potentials where large, transient depolarizing spontaneous fluctuations (DSFs) can approach AP threshold. This latent hyperactivity persisted for at least 3 weeks, whereas behavioral indicators of affective pain - hindpaw guarding and increased avoidance of a noxious substrate in an operant conflict test - persisted for 1 week or less. An unexpected discovery was latent OA in neurons from thoracic DRGs that innervate dermatomes distant from the injured tissue. The most consistent electrophysiological alteration associated with OA was enhancement of DSFs. Potential in vivo functions of widespread, low-frequency nociceptor OA consistent with these and other findings are to amplify hyperalgesic priming and to drive anxiety-related hypervigilance.

neuroscience↗

bioRxiv2020

A new Dicistrovirus from soldier fly Inopus flavus (James) (Diptera: Stratiomyidae), a pest of sugarcane.

The native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. While adult soldier flies do not feed on sugarcane, larvae spend one to two-years underground feeding on roots, causing mechanical and systemic damage to crops (Saccharum officinarum L.) that impacts yield. Current measures of pest control commonly target above ground pests and are ineffective against solider fly larvae, highlighting the importance of novel control methods. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the Dicistroviridae family. Viruses from this family have been found naturally infecting insects from a range of taxonomic groups and they often cause pathogenesis in their hosts. To characterise the genetic and physical properties of the new virus, the positive RNA genome was analysed using a combination of sequencing approaches. The virus genome is organised similarly to members of the Dicistroviridae with two open reading frames (ORF) the first encoding non-structural proteins and the second encoding structural proteins. The genome includes two potential internal ribosomal entry sites (IRES) one within the 5 UTR and the other in the intergenic region (IGR). Based on the amino acid sequences of the non-structural and structural polyproteins encoded by the two ORF soldier fly virus groups within the dicistrovirus family. Virus particles purified from infected larvae and visualised by electron microscopy are icosahedral, non-enveloped, and 30 nm in diameter. The genetic and physical characteristics of this novel soldier fly virus are consistent with it being a member of the Dicistroviridae.

microbiology↗

bioRxiv2019

VAMP8 contributes to TRIM6-mediated type-I interferon antiviral response during West Nile virus infection

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate hosts IFN-I response, West Nile Virus (WNV) replication is sensitive to pre-treatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNVs sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells WNV replication is significantly increased and IFN-I induction and signaling is impaired compared to wild-type (wt) cells. IFN{beta} pre-treatment was more effective in protecting against subsequent WNV infection in wt cells as compared to TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFN{beta} treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function is dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system.\n\nIMPORTANCEWNV is a mosquito-borne flavivirus that poses threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and viral-targeted antiviral strategies.

immunology↗