Biology subjects

McCormick, C.

Publications and source records attributed to McCormick, C..

8 recordsLinked to original sources

bioRxiv2018

The influenza A virus endoribonuclease PA-X usurps host mRNA processing machinery to limit host gene expression

Many viruses globally shut off host gene expression to inhibit activation of cell-intrinsic antiviral responses. However, host shutoff is not indiscriminate, since viral proteins and host proteins required for viral replication are still synthesized during shutoff. The molecular determinants of target selectivity in host shutoff remain incompletely understood. Here, we report that the influenza A virus shutoff factor PA-X usurps RNA splicing to selectively target host RNAs for destruction. PA-X preferentially degrades spliced mRNAs, both transcriptome-wide and in reporter assays. Moreover, proximity-labeling proteomics revealed that PA-X interacts with cellular proteins involved in RNA splicing. The interaction with splicing contributes to target discrimination and is unique among viral host shutoff nucleases. This novel mechanism sheds light on the specificity of viral control of host gene expression and may provide opportunities for development of new host-targeted antivirals.

microbiology↗

bioRxiv2018

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby displacement of the ER chaperone BiP activates UPR sensor proteins ATF6, PERK and IRE1 to initiate transcriptional responses to increase catabolic processes and ER folding capacity, while suppressing bulk protein synthesis. Kaposis sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, whereby the IRE1 endoribonuclease cleaves XBP1 mRNA, resulting in a ribosomal frameshift that yields the XBP1s transcription factor that transactivates the promoter of K-RTA, the viral lytic switch protein. By incorporating XBP1s responsive elements in the K-RTA promoter KSHV appears to have evolved a mechanism to respond to ER stress. Here, we report that following reactivation from latency, KSHV lytic replication causes activation of ATF6, PERK and IRE1 UPR sensor proteins. UPR sensor activation is required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminishes virion production. Despite strong UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to release the ATF6(N) transcription factor but known ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2 but ATF4 did not accumulate as expected; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein failed to accumulate and XBP1s-responsive genes were not transcribed. Remarkably, complementation of XBP1s deficiency during KSHV lytic replication by ectopic expression inhibited the production of infectious virions in a dose-dependent manner. Therefore, while XBP1s plays an important role in reactivation from latency, it inhibits later steps in lytic replication, which the virus overcomes by preventing its synthesis. Taken together, these findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.\n\nAuthor summaryHuman herpesvirus-8 is the most recently discovered human herpesvirus, and it is the infectious cause of Kaposis sarcoma, which is why its also known as Kaposis sarcoma-associated herpesvirus (KSHV). Like all herpesviruses, KSHV replicates in the cell nucleus and uses host cell machinery to convert viral genes into proteins. Some of these proteins are synthesized, folded and modified in the endoplasmic reticulum (ER) and traverse the cellular secretory apparatus. Because the virus heavily utilizes the ER to make and process proteins, there is potential to overwhelm the system, which could impede viral replication and in extreme cases, kill the cell. Normally, when demands on the protein folding machinery are exceeded then misfolded proteins accumulate and activate the unfolded protein response (UPR). The UPR resolves ER stress by putting the brakes on synthesis of many proteins, while signaling to the nucleus to turn on a program that aims to correct this imbalance. Previous work has shown that KSHV is wired to sense ER stress, which it uses to reactivate from a largely inactive state known as latency, in order to make more viruses. Specifically, a UPR sensor protein called IRE1 senses the accumulation of unfolded proteins in the ER and rededicates a gene called XBP1 to the production of a transcription factor called XBP1s through an unconventional cytoplasmic mRNA splicing event. XBP1s travels to the cell nucleus and stimulates the production of a collection of proteins that mitigate ER stress. In latently infected cells, XBP1s also binds to the KSHV genome and causes the production of K-RTA, a viral transcription factor that initiates the switch from latency to productive lytic replication. This achieves stress-induced initiation of KSHV replication, but nothing is known about how ER stress and the UPR affect progress through the KSHV replication cycle. Here we show that as KSHV replication progresses, all three known UPR sensor proteins, IRE1, ATF6 and PERK, are activated, which is required for efficient viral replication. Normally, activation of each of these three sensor proteins communicates a unique signal to the cell nucleus to stimulate the production of ER stress mitigating proteins, but in KSHV lytic replication all downstream communication is stymied. The failure to resolve ER stress would normally be expected to put the virus at a disadvantage, but we demonstrate that reversal of this scenario is worse; when we add extra XBP1s to the system to artificially stimulate the production of UPR responsive genes, virus replication is blocked at a late stage and no progeny viruses are released from infected cells. Taken together, these observations suggest that KSHV requires UPR sensor protein activation to replicate but has dramatically altered the outcome to prevent the synthesis of new UPR proteins and sustain stress in the ER compartment.

microbiology↗

bioRxiv2018

Differences in functional connectivity along the anterior-posterior axis of human hippocampal subfields

There is a paucity of information about how human hippocampal subfields are functionally connected to each other and to neighbouring extra-hippocampal cortices. In particular, little is known about whether patterns of functional connectivity (FC) differ down the anterior-posterior axis of each subfield. Here, using high resolution structural MRI we delineated the hippocampal subfields in healthy young adults. This included the CA fields, separating DG/CA4 from CA3, separating the pre/parasubiculum from the subiculum, and also segmenting the uncus. We then used high resolution resting state functional MRI to interrogate FC. We first analysed the FC of each hippocampal subfield in its entirety, in terms of FC with other subfields and with the neighbouring regions, namely entorhinal, perirhinal, posterior parahippocampal and retrosplenial cortices. Next, we analysed FC for different portions of each hippocampal subfield along its anterior-posterior axis, in terms of FC between different parts of a subfield, FC with other subfield portions, and FC of each subfield portion with the neighbouring cortical regions of interest. We found that intrinsic functional connectivity between the subfields aligned generally with the tri-synaptic circuit but also extended beyond it. Our findings also revealed that patterns of functional connectivity between the subfields and neighbouring cortical areas differed markedly along the anterior-posterior axis of each hippocampal subfield. Overall, these results contribute to ongoing efforts to characterise human hippocampal subfield connectivity, with implications for understanding hippocampal function. HighlightsO_LIHigh resolution resting state functional MRI scans were collected C_LIO_LIWe investigated functional connectivity (FC) of human hippocampal subfields C_LIO_LIWe specifically examined FC along the anterior-posterior axis of subfields C_LIO_LIFC between subfields extended beyond the canonical tri-synaptic circuit C_LIO_LIDifferent portions of subfields showed different patterns of FC with neocortex C_LI

neuroscience↗

bioRxiv2018

mTORC1 activity is dispensable for synthesis of KSHV lytic proteins

Herpesvirus genomes are decoded by host RNA polymerase II, generating viral messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5{square}-m7GTP caps and poly(A) tails that enable assembly of host translation initiation factors (TIFs) required for viral protein synthesis. Here we show that maximal production of viral proteins during lytic replication requires both the canonical host TIF eukaryotic initiation factor 4F (eIF4F) and the alternative host TIF eIF3d. Despite this, production of infectious virions is largely dependent on eIF3d, with eIF4F playing a minor role. Investigating the effects of eIF3d silencing on the host proteome revealed that it is required for accumulation of proteins involved in cholesterol metabolism, as well as the accumulation of intracellular cholesterol during KSHV lytic replication. In KSHV infected and uninfected cells alike, eIF3d supports efficient translation of cholesterol biosynthesis enzymes, including squalene epoxidase (SQLE), whose function is required to support productive lytic replication during cholesterol scarcity. These findings position eIF3d as a critical TIF required to support translation of essential host mRNAs required for KSHV replication. AUTHOR SUMMARYAnimal cells tightly regulate uptake, synthesis, storage, and export of cholesterol to ensure optimal membrane fluidity and permeability. Many enveloped viruses require cholesterol to support membrane fusion events during cell entry and cell surface budding events during assembly and egress. Herpesviruses like Kaposis sarcoma-associated virus (KSHV) require cholesterol to support entry, but its role in post-entry events, including budding and fusion at internal membranes, remains incompletely understood. Here, through our studies of KSHV protein synthesis, we uncovered a new mechanism of cellular control of cholesterol metabolism involving the host translation initiation factor (TIF) eIF3d. We demonstrated that eIF3d was required to support efficient translation of host mRNAs encoding key proteins required for cholesterol biosynthesis and uptake. Using a lytic reactivation model that bypasses the entry step, we observed that productive KSHV lytic replication is accompanied by eIF3d-dependent increases in intracellular cholesterol. Preventing cholesterol synthesis and uptake, or silencing eIF3d, strongly inhibited production of infectious virions. These studies shed new light on the importance of intracellular cholesterol to support KSHV replication, and provide motivation for future studies of how cholesterol influences discrete post-entry steps of KSHV infection.

microbiology↗

bioRxiv2018

Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a {beta}-xylanase, endoxylanase, {beta}-glucosidase, and an -L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative {beta}-xylanase into the pET26b(+) plasmid, enabling inducible gene expression in Escherichia coli (E. coli) and periplasmic localization. We demonstrated IPTG-inducible accumulation of {beta}-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative {beta}-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway into E. coli to enable biofuel production from wood pulp feedstock.

synthetic biology↗

bioRxiv2017

Differentiable processing of objects, associations and scenes within the hippocampus

The hippocampus is known to be important for a range of cognitive functions including episodic memory, spatial navigation and future-thinking. Wide agreement on the exact nature of its contribution has proved elusive, with some theories emphasising associative processes and another proposing that scene construction is its primary role. To directly compare these accounts of hippocampal function, we devised a novel mental imagery paradigm where different tasks were closely matched for associative processing and mental construction, but either did or did not evoke scene representations, and we combined this with high resolution functional MRI. The results were striking in showing that differentiable parts of the hippocampus, along with distinct cortical regions, were recruited for scene construction or non-scene-evoking associative processing. The contrasting patterns of neural engagement could not be accounted for by differences in eye movements, mnemonic processing or the phenomenology of mental imagery. These results inform conceptual debates in the field by showing that the hippocampus does not seem to favour one type of process over another; it is not a story of exclusivity. Rather, there may be different circuits within the hippocampus, each associated with different cortical inputs, which become engaged depending on the nature of the stimuli and the task at hand. Overall, our findings emphasise the importance of considering the hippocampus as a heterogeneous structure, and that a focus on characterising how specific portions of the hippocampus interact with other brain regions may promote a better understanding of its role in cognition.\n\nSignificance statementThe hippocampus is known to be important for a range of cognitive functions including episodic memory, spatial navigation and future-thinking. Wide agreement on the exact nature of its contribution has proved elusive. Here we used a novel mental imagery paradigm and high resolution fMRI to compare accounts of hippocampal function that emphasise associative processes with a theory that proposes scene construction as a primary role. The results were striking in showing that differentiable parts of the hippocampus, along with distinct cortical regions, were recruited for scene construction or non-scene-evoking associative processing. We conclude that a greater emphasis on characterising how specific portions of the hippocampus interact with other brain regions may promote a better understanding of its role in cognition.\n\nAbbreviations

neuroscience↗

bioRxiv2017

Stress granule-inducing eukaryotic translation initiation factor 4A inhibitors block influenza A virus replication

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5 untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host protein synthesis machinery to prevent viral replication.\n\nIMPORTANCEInfluenza A virus (IAV) relies on cellular protein synthesis to decode viral messenger RNAs. Pateamine A and silvestrol are natural products that inactivate an essential protein synthesis protein known as eIF4A. Here we show that IAV is sensitive to these eIF4A inhibitor drugs. Treatment of infected cells with pateamine A or silvestrol prevented synthesis of viral proteins, viral genome replication and release of infectious virions. The irreversible eIF4A inhibitor pateamine A sustained long-term blockade of viral replication, whereas viral protein synthesis quickly resumed after silvestrol was removed from infected cells. Prolonged incubation of either infected or uninfected cells with these drugs induced the programmed cell death cascade called apoptosis. Our findings suggest that core components of the host protein synthesis machinery are viable targets for antiviral drug discovery. The most promising drug candidates should selectively block protein synthesis in infected cells without perturbing bystander uninfected cells.

microbiology↗

bioRxiv2017

Mind-wandering in people with hippocampal amnesia

Subjective inner experiences, such as mind-wandering, represent the fundaments of human cognition. While the precise function of mind-wandering is still debated, it is increasingly acknowledged to have influence across cognition on processes such as future planning, creative thinking and problem-solving, and even on depressive rumination and other mental health disorders. Recently, there has been important progress in characterizing mind-wandering and identifying the neural networks associated with it. Two prominent features of mind-wandering are mental time travel and visuo-spatial imagery, which are often associated with the hippocampus. People with amnesia arising from selective bilateral hippocampal damage ( hippocampal amnesia) cannot vividly recall events from their past, envision their future or imagine fictitious scenes. This raises the question of whether the hippocampus plays a causal role in mind-wandering and if so, in what way. Leveraging a unique opportunity to shadow people with hippocampal amnesia for several days, we examined, for the first time, what they thought about spontaneously, without direct task demands. We found that they engaged in as much mind-wandering as control participants. However, whereas controls thought flexibly and creatively about the past, present and future, imagining vivid visual scenes, hippocampal amnesia resulted in thoughts primarily about the present comprising verbally-mediated semantic knowledge. These findings expose the hippocampus as a key pillar in the neural architecture of mind-wandering and also reveal its impact beyond memory, placing it at the heart of human mental life.\n\nSignificance statementHumans tend to mind-wander about 30-50% of their waking time. Two prominent features of this pervasive form of thought are mental time travel and visuo-spatial imagery, which are often associated with the hippocampus. To examine whether the hippocampus plays a causal role in mind-wandering, we examined the frequency and phenomenology of mind-wandering in patients with selective bilateral hippocampal damage. We found that they engaged in as much mind-wandering as controls. However, hippocampal damage changed the form and content of mind-wandering from flexible, episodic, and scene-based to abstract, semanticized, and verbal. These findings expose the hippocampus as a key pillar in the neural architecture of mind-wandering and reveal its impact beyond memory, placing it at the heart of our mental life.

neuroscience↗