Biology subjects

Hardy, R. W.

Publications and source records attributed to Hardy, R. W..

7 recordsLinked to original sources

bioRxiv2026

Skeletons in the closet: The importance of actin in alphavirus replication

The ability of the bacterial endosymbiont Wolbachia pipientis to block arboviruses in its mosquito host may be impinged by host genetic variation, leading to reduced efficacy in field releases. Across a large collection of Drosophila lines carrying natural genetic variation, we found that viral replication varied greatly in the absence of Wolbachia. However, the introduction of the symbiont reduced viral load in each background to similar levels, near the limit of detection. Therefore, Wolbachia-mediated viral blocking is seemingly robust against host genetic background. A genome-wide association study harnessing the variation in the viral loads across the Wolbachia-free set identified rhoGAP18B and betaCOP as host factors that contribute to SINV replication; furthermore, the gene products of which seemingly interact with each other in the context of cytoskeletal dynamics. These results shed light on host requirements for SINV replication and suggest possible avenues by which Wolbachia may encroach upon them during blocking.

microbiology↗

bioRxiv2023

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered. IMPORTANCEThe detection of chemical modifications to RNA is a rapidly expanding field within epigenetics. Nanopore sequencing technology provides an attractive means of detecting these modifications directly on the RNA, but accurate modification predictions are dependent upon the software developed to interpret the sequencing results. One of these tools, Tombo, allows users to detect modifications using sequencing results from a single RNA sample. However, we find that this method falsely predicts modifications in a specific sequence context across a variety of RNA samples, including RNA that lacks modifications. Results from previous publications include predictions in human coronaviruses with this sequence context and should be reconsidered. Our results highlight the importance of using RNA modification detection tools with caution in the absence of a control RNA sample for comparison.

genomics↗

bioRxiv2021

Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropod

Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy to reduce the replication of mosquito-borne RNA viruses such as alphaviruses. We previously demonstrated the importance of a host cytosine methyltransferase, DNMT2, in Drosophila and viral RNA as a cellular target during pathogen-blocking. Here we report on the role of DNMT2 in Wolbachia-induced alphavirus inhibition in Aedes species. Expression of DNMT2 in mosquito tissues, including the salivary glands, is elevated upon virus infection. Notably, this is suppressed in Wolbachia-colonized animals, coincident with reduced virus replication and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells is proviral, increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity is dependent on its methyltransferase activity. Finally, examining the effects of Wolbachia on modifications of viral RNA by LC-MS show a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is a vital mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental alphavirus replication and transmission processes.

microbiology↗

bioRxiv2020

Adaptive evolution in DNMT2 supports its role in the dipteran immune response

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects like fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggest DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Evidence of adaptive evolution, in the form of positive selection, can often be found in genes that are engaged in conflict with pathogens like viruses. Here we identify and describe evidence of positive selection that has occurred at different times over the course of DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection, and find they are restricted to four distinct domains of the protein and likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Additionally, we describe the role of the Drosophila-specific host protein IPOD, in regulating the expression and/or function of fruit fly DNMT2. Finally, heterologous expression of these orthologs suggest that DNMT2s role as an antiviral is host dependent, indicating a requirement for additional host-specific factors. Collectively, our findings highlight the adaptive evolution of DNMT2 in Dipteran insects, underscoring its role as an important, albeit non-canonical, regulator of host-pathogen interactions in mosquitoes and fruit flies.

microbiology↗

bioRxiv2020

Wolbachia and virus alter the host transcriptome at the interface of nucleotide metabolism pathways

Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for Dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.

microbiology↗

bioRxiv2020

The oncometabolite L-2-hydroxyglutarate is a common product of Dipteran larval development

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), Dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of Dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

developmental biology↗

bioRxiv2020

Viral RNA is a target for Wolbachia-mediated pathogen blocking

The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia- mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naive mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission. AUTHORS SUMMARYViruses transmitted by arthropod vectors pose a significant global health risk. Incidence of diseases caused by these viruses can thus be reduced by implementing effective vector control strategies. This need is further exacerbated due to the lack of commercially available vaccines and antivirals. Presence of the intracellular bacteria Wolbachia pipientis is associated with virus inhibition in multiple mosquito vectors. Furthermore, Wolbachia is inherited transovarially and spreads across the vector population like a natural gene drive, making it an attractive vector control agent. In this study, we examine how the presence of the bacterium in arthropod cells prevents initial establishment of vertebrate cell derived viruses. Our results indicate rapid turnover of incoming viral RNA very early during infection in Wolbachia-colonized cells, thus establishing it as a cellular target for pathogen blocking. Additionally, upon evaluating how these events might further limit virus spread, we find that infectivity of progeny viruses belonging to multiple RNA virus families are reduced on a per-particle basis. This aspect of virus inhibition is independent of any particular Wolbachia-host association and affects how these viruses replicate in naive mosquito and vertebrate cells, thus providing a collective basis of reduced virus dissemination and transmission in Wolbachia-colonized mosquitoes.

microbiology↗