Biology subjects

Pringle, E. S.

Publications and source records attributed to Pringle, E. S..

8 recordsLinked to original sources

bioRxiv2025

Stimulation of cholesterol efflux inhibits herpesvirus nuclear egress

Cholesterol in herpesvirus envelopes and host membranes supports membrane fusion during virus entry. However, little is known about how cholesterol affects other aspects of the viral replication cycle. Here, using an infection model that begins with reactivation from latency, bypassing the entry step, we demonstrate that depleting intracellular cholesterol inhibits the production of infectious Kaposis sarcoma-associated herpesvirus (KSHV). Treatment of latently infected cells with the liver X receptor (LXR) agonist 22(R)-hydroxycholesterol (22OH) increased expression of sterol response genes, including the cholesterol efflux pump ATP binding cassette subfamily A member 1 (ABCA1), and reduced intracellular cholesterol. 22OH treatment initially enhanced lytic reactivation, but diminished viral protein accumulation during late replication, while altering accumulation of host proteins that regulate the cell cycle and innate immunity. Cholesterol efflux led to an increase in the proportion of capsids lacking viral genomes in the nucleus and a reduction in nucleocapsids accessing the cytoplasm. Next, we used a herpes simplex virus (HSV) infection model to explore if 22OH affected the conserved herpesvirus nuclear egress complex (NEC), comprised of two viral proteins that accumulate at the inner nuclear membrane and facilitate primary envelopment. We found that 22OH inhibited HSV-1 and HSV-2 replication and spread in plaque reduction assays and impaired localization of NEC proteins pUL31 and pUL34 to the nuclear periphery. These findings indicate that cholesterol efflux inhibits herpesvirus replication by interfering with the formation of nucleocapsids competent to complete nuclear egress. IMPORTANCECholesterol plays key roles in regulating the fluidity of cellular membranes and the trafficking and lateral diffusion of transmembrane proteins. Herpesviruses encode a variety of transmembrane proteins that traffic to different cellular compartments, while herpesvirus capsids and tegument proteins interact with nuclear and trans-Golgi network membranes throughout the primary and secondary envelopment process, respectively. This produces viral particles with cholesterol as an envelope component which is required to support membrane fusion during entry. Our discovery that cholesterol efflux inhibits KSHV nuclear egress suggests that cholesterol plays important supportive roles at this stage of lytic replication. Moreover, we observed that cholesterol efflux mediated by oxysterol treatment has widespread effects on host cell protein expression, resulting in a non-classical antiviral state. Cholesterol likely supports the proper trafficking of viral proteins to nuclear sites of virion assembly or is influencing the fluidity of nuclear membranes used for viral primary envelopment and de-envelopment during nuclear egress.

microbiology↗

bioRxiv2025

Coronavirus M proteins disperse the trans-Golgi network and inhibit anterograde protein trafficking in the secretory pathway

Coronaviruses (CoVs) encode Spike, Membrane (M), and Envelope (E) transmembrane proteins that are translated and processed at the endoplasmic reticulum (ER) and traverse the secretory pathway to converge at sites of virus assembly. Three transmembrane ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK), sense the accumulation of unfolded proteins in the ER and initiate the unfolded protein response (UPR) to increase ER protein folding capacity. We observed UPR modulation by numerous CoV proteins, including Spike, which broadly activated all three arms of the UPR. By contrast, M selectively inhibited the ATF6 arm of the UPR, either when it was activated by CoV proteins like Spike, or when it was activated by chemical stimuli of ER stress; however, M was unable to inhibit Spike-mediated activation of IRE1 or PERK. ATF6 inhibition was conserved amongst all human CoV M proteins. Amongst the UPR sensors, ATF6 has a unique activation mechanism whereby ER stress triggers translocation to the Golgi where ATF6 is processed by resident proteases to release the ATF6-N bZIP transcription factor. Because M had no effect on the function of the ATF6-N transcription factor, we reasoned that it may act upstream by inhibiting ATF6 trafficking in the secretory pathway. Indeed, we observed that ectopically expressed M inhibited several processes that require ER-to-Golgi transport, including sterol regulatory element binding protein-2 (SREBP2)-mediated activation of sterol responses and stimulator of interferon response cGAMP interactor 1 (STING1)-mediated activation of interferon responses. M also inhibited the secretion of a soluble Gaussia luciferase reporter protein. Using a Retention Using Selective Hooks (RUSH) cargo sorting assay, we observed that M accumulated in the cis-Golgi and inhibited further anterograde transport of a transmembrane reporter protein beyond this compartment, while dispersing the trans-Golgi network (TGN). We also observed a conserved TGN dispersal phenotype in cells infected with SARS-CoV-2, hCoV-OC43, or hCoV-229E. We determined that M is present in both detergent resistant and detergent soluble membranes and that M increased cholesterol abundance at the cis-Golgi. Together, these observations suggest that CoV M proteins disrupt the TGN and impede normal anterograde traffic in the canonical secretory pathway, potentially by increasing cholesterol levels at the cis-Golgi . Because CoV egress does not require the TGN, this mechanism could allow the virus to selectively interfere with host responses to infection without impeding egress of nascent virions. IMPORTANCEMany viruses encode proteins that limit host antiviral responses. Coronaviruses encode a remarkably diverse array of proteins that antagonize host responses at discrete steps including the detection of viral products, signal transduction, host mRNA processing and nuclear export, and protein synthesis. Here, we describe a new form of viral antagonism of host antiviral responses by remodelling the secretory pathway, dispersing a distal portion of the Golgi network and causing accumulation of proteins early in the Golgi. This is achieved by an abundant viral structural transmembrane glycoprotein M, which is best known for its role as the central player in the assembly of new viruses. This newly discovered function of M allows it to limit host antiviral responses that depend on trafficking in the late secretory pathway, while maintaining its role in virus assembly in the early secretory pathway.

microbiology↗

bioRxiv2024

A yeast-based reverse genetics system to generate HCoV-OC43 reporter viruses encoding an eighth sgRNA

Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Here, we report the development of a new reverse genetics system for the endemic human coronavirus (HCoV) OC43 that utilizes transformation-associated recombination (TAR) to assemble complete viral genomes from dsDNA genome fragments via homologous recombination in Saccharomyces cerevisiae. Following cDNA synthesis from HCoV-OC43 viral RNA, we used TAR to capture fragments of the HCoV-OC43 genome to store as sequence-validated dsDNA parts. We performed combinatorial assembly in yeast to obtain an intact dsDNA copy of the HCoV-OC43 genome sufficient to launch viral replication upon introduction into human cells, yielding the yeast assembled OC43YA virus. We also expanded the OC43YA genome by inserting an eighth body transcription regulatory sequence (B-TRS) and an mClover3-H2B reporter gene between the M and N genes, designed to allow the reporter protein to be translated from its own subgenomic mRNA. We thoroughly evaluated OC43YA and the OC43-mCloYA reporter virus, and demonstrated comparable viral gene expression, fitness in cell culture, and susceptibility to antivirals, compared to their natural progenitor. In summary, this new HCoV-OC43 reverse genetics system provides a modular platform for mutagenesis and combinatorial assembly of HCoV-OC43 genomes, and demonstrates the feasibility of expanding the genome while avoiding disruption of native coding sequences. IMPORTANCEHuman coronavirus OC43 (HCoV-OC43) is an endemic human coronavirus that typically causes relatively mild respiratory illnesses and displays seasonal patterns of infection. We developed a new system to assemble DNA copies of HCoV-OC43 genomes and generate recombinant viruses for research purposes. This system uses yeast, first to capture segments of DNA encompassing the entire RNA-based viral genome, and then to stitch them together into complete DNA genome copies that can be amplified in bacteria and introduced into human cells to initiate an infectious cycle, ultimately yielding recombinant viruses with comparable properties to their natural progenitors. We also devised a strategy to expand the viral genome, adding a gene for a reporter protein encoded by an additional eighth subgenomic mRNA. This yeast-based genome assembly system provides a modular platform for rapid mutagenesis and combinatorial assembly of HCoV-OC43 genomes and demonstrates the feasibility of expanding the genome.

synthetic biology↗

bioRxiv2023

Dual inhibition of coronavirus Mpro and PLpro enzymes by phenothiazines and their antiviral activity

Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity in vitro was identified. In silico docking studies also predicted binding of the phenothiazine to the active sites of Mpro and PLpro from distantly related alphacoronavirus, HCoV-229E (229E) and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cell culture infection models. It was further demonstrated that the compound inhibited 229E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/557219v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@fa2c92org.highwire.dtl.DTLVardef@844484org.highwire.dtl.DTLVardef@1ae5d5aorg.highwire.dtl.DTLVardef@730133_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICoronavirus cysteine proteases Mpro and PLpro are targets for novel antiviral agents C_LIO_LIPhenothiazine ureas inhibit SARS-CoV-2 Mpro and PLpro protease activity C_LIO_LISome phenothiazine ureas inhibit replication of diverse coronaviruses with minimal cytotoxicity C_LIO_LIPhenothiazine ureas inhibit early stages of coronavirus replication consistent with failure of viral polyprotein cleavage C_LI

microbiology↗

bioRxiv2022

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2, and to a lesser extent, the alphacoronavirus HCoV-229E. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting synthesis of full-length and subgenomic HCoV RNAs. Furthermore, consistent with our previous report on the effects of thiopurines on influenza A virus glycoproteins, we observed that 6-TG inhibited accumulation of Spike glycoproteins from diverse HCoVs. Specifically, 6-TG treatment decreased the accumulation of Spike proteins and increased their electrophoretic mobility, consistent with Spike migration following the enzymatic removal of N-linked oligosaccharides with Peptide:N-glycosidase F (PNGaseF). SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on lentiviruses pseudotyped with SARS-CoV-2 Spike; lentiviruses could be harvested from cell supernatants but were deficient in Spike and unable to infect human cells bearing ACE2 receptors. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. At low micromolar doses, the primary known mode of action of 6-TG is selective inhibition of the small GTPase Rac1. However, we observed that selective chemical inhibitors of the small GTPases Rac1, CDC42 and Rho had no effect on Spike processing and accumulation. The GTPase agonist ML099 countered the effects of 6-TG, suggesting that an unknown GTPase could be the relevant 6-TG-target protein involved in regulating Spike processing and accumulation. Overall, these findings provide important clues about the mechanism of action of a candidate antiviral that can broadly target HCoVs and suggest that small GTPases are promising targets for host-targeted antivirals. AUTHOR SUMMARYThe COVID-19 pandemic has ignited efforts to repurpose existing drugs as safe and effective antivirals. Rather than directly inhibiting viral enzymes, host-targeted antivirals inhibit host cell processes to indirectly impede viral replication and/or stimulate antiviral responses. Here, we describe a new antiviral mechanism of action for the FDA-approved thiopurine 6-thioguanine. We demonstrate that this thiopurine is a pro-drug that must be metabolized by host enzymes to gain antiviral activity. We show that it can inhibit the replication of several human coronaviruses, including SARS-CoV-2, at least in part by interfering with the processing and accumulation of Spike glycoproteins, thereby impeding assembly of infectious progeny viruses. We provide evidence implicating host cell GTPase enzymes in the antiviral mechanism of action.

microbiology↗

bioRxiv2021

A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

Kaposis sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposis sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the "K-genes", are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. Kaposin-deficient latent iSLK cell lines displayed reduced viral genome copy number and often exhibited small LANA nuclear bodies; despite this, all were capable of progeny virion production. Primary infection with {Delta}KapB virus revealed decreased LANA expression and viral genome copy number, yet providing KapB protein in trans failed to complement these defects, suggesting a requirement for the kaposin locus in cis. Our previous work showed that KapB was sufficient to recapitulate the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). We now show that KapB is necessary for PB disassembly during latent KSHV infection. These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci. ImportanceKaposis sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.

microbiology↗

bioRxiv2020

Thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein accumulation in cell culture infection model

Enveloped viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), utilize the host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. Using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that selectively disrupted the processing and accumulation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA). Selective disruption of IAV glycoprotein processing and accumulation by 6-TG and 6-TGo correlated with unfolded protein response (UPR) activation and HA accumulation could be partially restored by the chemical chaperone 4-phenylbutyrate (4PBA). Chemical inhibition of the integrated stress response (ISR) restored accumulation of NA monomers in the presence of 6-TG or 6-TGo, but did not restore NA glycosylation or oligomerization. Thiopurines inhibited replication of the human coronavirus OC43 (HCoV-OC43), which also correlated with UPR/ISR activation and diminished accumulation of ORF1ab and nucleocapsid (N) mRNAs and N protein, which suggests broader disruption of coronavirus gene expression in ER-derived cytoplasmic compartments. The chemically similar thiopurine 6-mercaptopurine (6-MP) had little effect on the UPR and did not affect IAV or HCoV-OC43 replication. Consistent with reports on other CoV Spike (S) proteins, ectopic expression of SARS-CoV-2 S protein caused UPR activation. 6-TG treatment inhibited accumulation of full length S0 or furin-cleaved S2 fusion proteins, but spared the S1 ectodomain. DBeQ, which inhibits the p97 AAA-ATPase required for retrotranslocation of ubiquitinated misfolded proteins during ER-associated degradation (ERAD) restored accumulation of S0 and S2 proteins in the presence of 6-TG, suggesting that 6-TG induced UPR accelerates ERAD-mediated turnover of membrane-anchored S0 and S2 glycoproteins. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and disrupt accumulation of viral glycoproteins. Importantly, our data demonstrate for the first time the efficacy of these thiopurines in limiting IAV and HCoV-OC43 replication in cell culture models. IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. During infection, many viruses burden the ER with the task of creating and processing viral glycoproteins that will ultimately be incorporated into viral envelopes. Some viruses refashion the ER into replication compartments where viral gene expression and genome replication take place. This viral burden on the ER can trigger the cellular unfolded protein response (UPR), which attempts to increase the protein folding and processing capacity of the ER to match the protein load. Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR in a manner that impedes viral glycoprotein accumulation for enveloped influenza viruses and coronaviruses. These drugs may impede the replication of viruses that require precise tuning of the UPR to support viral glycoprotein synthesis for the successful completion of a replication cycle.

microbiology↗

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↗