Biology subjects

Spear, E.

Publications and source records attributed to Spear, E..

5 recordsLinked to original sources

bioRxiv2026

HIGH DOSE GAMMAHERPESVIRUS INFECTION IN MACROPHAGES RESULTS IN INFLAMMATORY MULTIMODAL CELL DEATH

Human gammaherpesviruses (gHVs) including Epstein-Barr Virus and Kaposis sarcoma associated herpesvirus are linked to cancer and inflammatory disease development. The outcome of gHV infection is highly regulated by cell type, with macrophages identified as an important early infection target that can also serve as a latency reservoir. Here, we used the mouse gHV model, murine gammaherpesvirus (MHV68), to identify a dose-dependent outcome of MHV68 infection of macrophages, with high dose infection resulting in extensive morphological changes, viral gene expression, and a pronounced inflammatory cell death not observed under conditions of lytic replication occurring at lower infectious dose. Virion envelope and tegument components were not sufficient for this dose-dependent cell death, which required intact viral DNA and virus transcription, yet occurred independently of viral DNA replication and late gene transcription. Necrostatin-1 (Nec-1) treatment, an anti-inflammatory therapeutic, limited cell death and reduced cell morphology alterations, with no impact on virus replication, uncoupling cell death from lytic replication. Activation of autophagy profoundly limited high dose infection outcomes, with increased cell survival and reduced viral gene expression. Despite the improved cell survival observed in cells treated with Nec-1, biochemical analysis of infected cultures identified robust apoptosis induction with subsequent activation of gasdermin E, suggesting a multi-modal cell death mechanism culminating in pyroptosis. Our findings suggest that high dose macrophage infection byMHV68 is a major driver of inflammatory cell death to potentially shape downstream inflammation and immune responses.

microbiology↗

bioRxiv2026

An A-rich linker between dengue virus tandem xrRNAs facilitates functional coordination

Orthoflaviviruses use programmed resistance to host 5' to 3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs) during infection. This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures that often occur in tandem and whose function can be coupled. In dengue virus serotype 2 (DENV2) this coupling results in changing patterns of sfRNA identity and abundance linked to the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryoEM map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings provide an explanation for the coupling between tandem xrRNAs and suggests new mechanistic hypotheses. IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2 this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.

microbiology↗

bioRxiv2024

Oomycete communities in lowland tropical forest soils vary in abundance and are composed of saprophytes and pathogens of seeds and seedlings of multiple plant species.

PremiseThe soils in lowland tropics are teeming with microbial life which can impact plant community structure and diversity through plant-soil feedbacks. While bacteria and fungi have been the focus of most studies in the tropics, the oomycetes may have an outsized effect on seed and seedling health and survival, given their affinity for environments with increased precipitation and temperature. MethodsWe assessed the diversity and pathogenicity of oomycete species present in a lowland tropical forest in Panama. We used both a culture dependent leaf-baiting assay and culture independent soil DNA metabarcoding methods to quantify zoospore abundance and species diversity. A subset of the isolates from the baiting assay were used to evaluate pathogenicity and aggressiveness on seedlings of three tree species. Key resultsOomycetes are ubiquitous and common members of the soil microbial community in lowland tropical forests and zoospore abundance was far greater compared to similar studies from temperate and mediterranean forests. We also observed variation in oomycete species ability to infect host plants. Species of Pythium were more aggressive, while species of Phytopythium caused less disease but were more diverse and commonly isolated from the soil. Finally, we found that individual hosts accumulate a distinct oomycete community and was the only factor that had an effect community structure. ConclusionsCollectively, these finding demonstrate that oomycetes are ubiquitous, host-generalist pathogens and saprophytes, that have the potential to impact seed and seedling survival in lowland tropical forests

ecology↗

bioRxiv2020

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Human ZMPSTE24, an integral membrane zinc metalloprotease, is required for conversion of prelamin A to mature lamin A, a component of the nuclear lamina and failure of this processing causes premature ageing disorders. ZMPSTE24 has also been implicated in both type 2 diabetes mellitus and in viral-host response mechanisms, but to date its only confirmed substrate is the precursor for lamin A. Prelamin A is thought to undergo four C-terminal post-translational modifications in the following order: farnesylation, SIM tripeptide cleavage, carboxymethylation and upstream "SY^LL" cleavage. Here we present evidence that the sequence of events does not follow the accepted dogma. We assessed cleavage of long human prelamin A sequence peptides by purified human ZMPSTE24 combined with FRET and mass spectrometry to detect products. Surprisingly, we found that the "SY^LL" cleavage occurs before and independent of the C-terminal CSIM modifications. We also found that ZMPSTE24 does not perform the predicted C^SIM tripeptide cleavage, but rather it removes an IM dipeptide. ZMPSTE24 can perform a tripeptide cleavage with a canonical CaaX box (C: cysteine; a: aliphatic; X: any residue), but the C-terminus of prelamin A is not a true CaaX sequence. Regardless of the C-terminal modifications of prelamin A, ZMPSTE24 can perform upstream SY^LL cleavage, thus removing the unwanted farnesylated C-terminus. Therefore, it is failure of SY^LL cleavage, not the C-terminal processing that is the likely cause of progeroid disorders.

biochemistry↗

bioRxiv2017

ZMPSTE24 Missense Mutations that Cause Progeroid Diseases Decrease Prelamin A Cleavage Activity, Protein Stability, or Both

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein critical for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA. After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor, prelamin A, undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and the related progeroid disorders mandibuloacral dysplasia-type B (MAD-B) and restrictive dermopathy (RD). Here we report a \"humanized yeast\" system to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability, or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in ER-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib, which may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this \"humanized yeast system\" can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

cell biology↗