Biology subjects

Duguay, B.

Publications and source records attributed to Duguay, B..

5 recordsLinked to original sources

bioRxiv2026

Herpes simplex virus 1 subverts the mitochondrial network to support the infection: A lesson on mitochondrial versatility

Herpes simplex virus 1 (HSV-1) infects approximately 67% of the population worldwide. It establishes lifelong reservoirs in sensory neurons and has been linked to several diseases including neuronal dysfunction. Disruption of mitochondrial homeostasis is a hallmark of HSV-1 infection, however a molecular understanding of these changes and their significance is not yet well defined. HSV-1 infection causes a UL12.5-dependent inhibition of mitochondrial biogenesis through the loss of mitochondrial DNA and mitochondrial transcription factors, PGC-1 (peroxisome proliferator-activated receptor-gamma co-activator) and TFAM (mitochondrial transcription factor). Conversely, UL12.5-independent mechanisms inhibit mitochondrial fusion by activating the OMA1 metallopeptidase that cleaves the inner mitochondrial membrane fusion protein OPA1 (optic atrophy protein 1) and by down-modulating the outer mitochondrial membrane fusion protein MFN2 (mitofusin 2). This inhibition of fusion results in a smaller mitochondrial network that clusters to perinuclear regions, likely supplying energy for viral replication and envelopment. The inner mitochondrial membrane protein TIM23 is also down-modulated during infection in a UL12.5-independent mechanism. Failure of the virus to promote these changes negatively impacts the infection. Despite these changes, mitochondria are protected from mitophagy due to the viral-induced degradation of several mitophagy adaptor proteins, whereby damaged mitochondrial components, including mitochondrial DNA, are extruded via extracellular vesicles. These mitochondrial changes still support functions necessary for HSV-1 infection. Basal cell respiration is preserved, while spare respiratory capacity and extracellular acidification rates increase, indicating glycolytic activity. Mitochondrial membrane potential is also preserved. Overall, our studies provide mechanistic insight into how HSV-1 impacts mitochondria, which could contribute to viral pathogenesis.

microbiology↗

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↗

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↗

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↗