Biology subjects

Poeschla, E. M.

Publications and source records attributed to Poeschla, E. M..

6 recordsLinked to original sources

bioRxiv2025

ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

Sensing of viral double-stranded RNA by MDA5 triggers abundant but transient interferon-stimulated gene (ISGs) expression. If dsRNA synthesis is made persistent by transgenically expressing a picornaviral RNA-dependent RNA polymerase (RdRp) in mice, lifelong MDA5-MAVS pathway activation and marked, global ISG upregulation result. This confers robust protection from viral diseases but in contrast to numerous other chronic MDA5 hyperactivation states, the mice suffer no autoimmune or other health consequences. Here we find they further confound expectations by being resistant to a strong autoimmunity (lupus) provocation. However, knockout of one allele of Adar breaks the autoinflammation-protected state of RdRptg mice and results in a severe disease that resembles interferonopathies caused by MDA5 gain-of-function protein mutations. Adar+/- mice are healthy but Adar+/- RdRptg mice have shortened lifespan, stunted growth, premature fur graying, poorly developed teeth, skeletal abnormalities, and extreme ISG elevations. A-to-I edits are both abnormally distributed and increased (numbers of genes and sites). These results, with a nucleic acid-triggered and MDA5-wild type model, illuminate the ADAR1-MDA5 axis in the regulation of innate immunity and establish that viral polymerase-sourced dsRNA can drive autoinflammatory disease pathogenesis. IMPORTANCERNA virus double-stranded RNAs are important pathogen associated molecular patterns that are sensed by the RIG-I like receptor MDA5, which triggers an acute innate immune response involving many ISGs. One key to a healthy innate immune system is that MDA5 not sense endogenous dsRNA. This is normally ensured by dsRNA duplex-disrupting ADAR1 editing of host dsRNAs. Picornavirus RdRptg mice have an unusual constitutive MDA5 activation state, with very high lifelong MDA5-mediated ISG expression that confers robust protection from diverse lethal viruses. Importantly, and in contrast to numerous other chronic MDA5 hyperactivation states, the mice develop no autoinflammatory consequences. If we delete one ADAR1 allele, however, which by itself is well tolerated, the mice develop a multisystem disease that resembles the human interferonopathy Singleton-Merten syndrome. In contrast to other MDA5/ADAR1 disease models, the MDA5 and ADAR1 proteins are both wild type in this dsRNA-driven model.

immunology↗

bioRxiv2024

Disruption of LEDGF/p75-directed integration derepresses antisensetranscription of the HIV-1 genome

AbstractDisruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

microbiology↗

bioRxiv2022

Multivalent interactions essential for lentiviral integrase function

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits (1). Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 [A] resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the -helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching revealed that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 resulted in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

biochemistry↗

bioRxiv2021

A Lentiviral Envelope Signal Sequence is a Tetherin Antagonizing Protein

Signal sequences are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus Feline immunodeficiency virus (FIV) contains the longest signal sequence of all eukaryotic, prokaryotic and viral proteins (175 amino acids). The reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here we identify the FIV Env signal sequence (Fess) as the FIV tetherin antagonist. A short deletion in the central portion of Fess had no effect on viral replication in the absence of tetherin but severely impaired virion budding in its presence. Fess is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, it functions by stringently blocking the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane.

microbiology↗

bioRxiv2019

dsRNA-Seq: Identification of viral infection by purifying and sequencing dsRNA

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that all RNA viruses produce dsRNA while replicating. Purifying and sequencing dsRNA from total RNA isolated from infected tissue allowed us to recover replicating viral sequences. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length RNA viruses of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that dont have sequenced genomes and/or commercially available rRNA depletion reagents. Similar to other metagenomic strategies, dsRNA-Seq has the potential to identify unknown viral disease agents that share little to no similarity to known viruses. However, the significant advantage of this method is the ability to identify replicated viral sequences, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

molecular biology↗

bioRxiv2017

Characterization Of Vif Domains That Mediate Feline Immunodeficiency Virus Antagonism Of APOBEC3-H And APOBEC3-CH Restriction

Feline immunodeficiency virus (FIV) Vif mediates degradation of two anti-lentiviral feline APOBEC3 (fA3) proteins, fA3Z3 and fA3Z2bZ3. HIV-1 Vif targets the restriction factor human APOBEC3G (A3G, hA3Z2g-Z1c) for proteasome degradation to mediate viral evasion. Despite this similarity, FIV and HIV-1 Vif share limited homology. Vif binds hA3Z2g-Z1c through its N-terminal region, while its C-terminal region binds to an E3-ligase complex containing Cullin5 and Elongin B/C. Further, HIV-1 Vif contains critical domains in its C-terminus, including an adjacent BC box, the only shared domain between FIV and HIV-1 Vif, and a non-classical zinc finger (HCCH) domain. Felid lentivirus Vif, however, contains a highly conserved KCCC motif. While both Vifs have evolved to counteract select A3 antiretroviral proteins, the FIV Vif domains necessary to target fA3s for degradation are incompletely understood. To identify these domains, we used the well-characterized HIV-1 Vif domains to show that distinct mutations within the BC box of FIV Vif prevent fA3Z3 and fA3Z2bZ3 degradation and reduce virion infectivity. We also found that mutating any single residue in the KCCC motif blocked fA3 targeting and impaired FIV infectivity and replication. These mutations also failed to disrupt the FIV Vif and Cullin5 interaction. Further, we showed that, in contrast to the HCCH domain in HIV-1 Vif, the KCCC domain of FIV Vif does not bind zinc. However, unlike HIV-1 Vif, FIV Vif (C36 isolate) reduces intracellular levels of co-expressed Cullin5 proteins, a novel finding. Our results reveal important C-terminal residues in FIV Vif and show that the BC box and KCCC regions are critical for fA3 degradation, infectivity, and spreading replication.

immunology↗