Biology subjects

Kistler, A. L.

Publications and source records attributed to Kistler, A. L..

6 recordsLinked to original sources

bioRxiv2025

Replicating without stress: a dengue replicon model reveals distinct host rewiring to accommodate persistence

Self-replicating viral RNAs, or replicons, are widely used to study virus-host interactions and screen antiviral compounds, yet their ability to model persistent infection and host adaptation remains poorly understood. Here, we characterize a stable subgenomic dengue virus serotype 2 (DENV2) replicon (DENV2-rep) that supports long-term viral RNA replication in human hepatoma cells without triggering cytotoxicity or canonical stress responses. Compared to live DENV2 infection, which induces ER stress and global translation shutdown, DENV2-rep cells maintain active translation and polysome integrity, while mounting an antiviral transcriptional response and resisting superinfection. Transcriptomic and proteomic profiling reveals that DENV2-rep cells accommodate long-term replication by remodeling of metabolic, secretory, and cytoskeletal pathways. In contrast, acute infection uncouples transcription from protein synthesis, limiting host proteome remodeling. We also detected marked depletion of the NS5 RNA polymerase in the DENV2-rep context. Together, these findings highlight host pathways that can be rewired to support persistent RNA replication and uncover post-translational regulation of viral proteins as a potential vulnerability. Our work clarifies the strengths and limitations of replicons as models for infection and reveals how persistent vRNA replication is tolerated through selective remodeling of translation, organelle composition, and protein stability.

microbiology↗

bioRxiv2025

Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

Pooled CRISPR knockout (KO) screens using live viruses are a proven and valuable approach for identifying essential host factors acting across viral life cycles. Here we describe the development of a pooled genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically identify host factors essential for viral replication. Virus replicons are non-infectious, therefore enabling the study of highly virulent viruses under standard biosafety level 2 containment. We developed a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line to perform a pooled genome-wide FACS-based CRISPR KO screen. This benchmark DENV-2 replicon screen successfully identified host genes previously known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components) and confirmed two additional genes (DOHH and ZFP36L2) involved in replication that have not been recovered in prior live virus screens. We applied this replicon screening approach to two highly divergent viruses: chikungunya virus (CHIKV), a positive-sense RNA virus that replicates at the plasma membrane, and Ebola virus (EBOV), a negative-sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified two genes known to be required for replication (G3BP1 and G3BP2) and several additional, novel genes (CLEC4G, CSDE1, GOLGA7, HNF1A, and PCBD1). We verified two of them (CSDE1 and GOLGA7) in live CHIKV replication assays. A distinct set of genes (EHMT1, EHMT2, and USP7) were recovered in the EBOV replicon screen and were further confirmed using independent transient transfection assays. Thus, viral replicon-based screens provide a useful approach that can be extended to viruses of diverse taxa to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed medical countermeasures.

genomics↗

bioRxiv2024

Discovery of a novel bandavirus using metagenomic sequencing in a retrospective analysis of an unresolved 2020 mortality event involving black vultures in the northeastern United States.

Investigations of wildlife diseases and mortality events can sometimes lead to inconclusive results due to limitations in diagnostics combined with an ever-increasing number of emerging viruses. The use of tools such as unbiased metagenomic next generation sequencing (mNGS) can facilitate the identification of causative agents where conventional investigation methods fail. We performed a retrospective mNGS analysis on RNA isolated from postmortem samples collected during a black vulture (Coragyps atratus, family: Cathartidae) mortality event that occurred in eastern Pennsylvania and western New Jersey in 2020. We describe the discovery and identification of a novel species of bandavirus (Phenuiviridae family) in case specimens from this die-off, as well as some of the associated pathological findings. The Bandavirus genus comprises tickborne viral species that have been reported across five continents and implicated in outbreaks in a variety of mammalian hosts, including humans, and in avian species making them important potential sources of zoonotic spillover events. Genomic and phylogenetic analysis of the bandavirus detected in this study indicate its closest relative corresponds to Hunter Island virus, a bandavirus previously implicated in albatross mortality events off the coast of Tasmania, Australia. Follow-up PCR testing of samples from additional vultures from the same cohort confirmed that this new bandavirus is the likely cause of death.

genomics↗

bioRxiv2023

Detection, characterization, and phylogenetic analysis of a near-whole genome sequence of a novel astrovirus in an endemic Malagasy fruit bat, Rousettus madagascariensis

Bats (order: Chiroptera) are known to host a diverse range of viruses, some of which present a public health risk. Thorough viral surveillance is therefore essential to predict and potentially mitigate zoonotic spillover. Astroviruses (family: Astroviridae) are an understudied group of viruses with a growing amount of indirect evidence for zoonotic transfer. Astroviruses have been detected in bats with significant prevalence and diversity, suggesting that bats may act as important astrovirus hosts. Most astrovirus surveillance in wild bat hosts has, to date, been restricted to single-gene PCR detection and concomitant Sanger sequencing; additionally, many bat species and many geographic regions have not yet been surveyed for astroviruses at all. Here, we use metagenomic Next Generation Sequencing (mNGS) to detect astroviruses in three species of Madagascar fruit bats, Eidolon dupreanum, Pteropus rufus, and Rousettus madagascariensis. We detect numerous partial sequences from all three species and one near-full length astrovirus sequence from Rousettus madagascariensis, which we use to characterize the evolutionary history of astroviruses both within bats and the broader mammalian clade, Mamastrovirus. Taken together, applications of mNGS implicate bats as important astrovirus hosts and demonstrate novel patterns of bat astrovirus evolutionary history, particularly in the Southwest Indian Ocean region.

evolutionary biology↗

bioRxiv2021

Full genome Nobecovirus sequences from Malagasy fruit bats define a unique evolutionary history for this coronavirus clade

Bats are natural reservoirs for both Alpha- and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat (Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis. Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats.

evolutionary biology↗

bioRxiv2018

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

synthetic biology↗