Biology subjects

Colpitts, C. C.

Publications and source records attributed to Colpitts, C. C..

8 recordsLinked to original sources

bioRxiv2026

Hepatitis C virus NS5A is not an effective antagonist of the canonical PKR-eIF2α pathway

Protein kinase R (PKR) is an antiviral sensor that induces translational shutdown and stress granule (SG) formation through the phosphorylation of eIF2. Although previous studies have proposed that hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibits PKR dimerization, other studies consistently observe PKR activation and SG formation during HCV infection. To address this discrepancy, we interrogated the ability of NS5A from different genotypes and strains to regulate canonical PKR-eIF2 activation in a variety of cell culture models. Regardless of the genotype or strain tested, HCV NS5A failed to inhibit PKR dimerization or dsRNA-induced PKR activation, nor did it rescue downstream steps in the PKR-eIF2 pathway, such as translation halt and SG formation, in contrast to known viral PKR antagonists. Similarly, NS5A did not antagonise PKR activation when expressed in the context of other HCV non-structural proteins. We also tested the role of the host factor cyclophilin A (CypA), which interacts with both NS5A and PKR. However, the absence of CypA did not affect downstream PKR signaling or the activity of viral PKR antagonists or NS5A. Collectively, these findings indicate that NS5A does not effectively antagonise the PKR-eIF2 pathway and further support previous findings that HCV replication occurs in the presence of an active PKR response.

microbiology↗

bioRxiv2026

MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

Thapsigargin (Tg), an inducer of endoplasmic reticulum stress and the unfolded protein response (UPR), has broad-spectrum antiviral activity, although the underlying mechanisms remain unclear. Here, we characterized its antiviral mechanism(s) using hepatitis C virus (HCV) as a model. Pre-treatment with Tg partially inhibited HCV RNA replication, but strongly reduced extracellular viral titer and RNA, suggesting a block to later stages of infection. Silencing the expression of ATF6, PERK or IRE1 did not significantly impact the antiviral activity of Tg. Treatment with tunicamycin, which activates the UPR by a different mechanism, did not exert the same antiviral effect, indicating potential UPR-independent antiviral mechanisms for Tg. Given the importance of lipid droplets (LDs) and lipid metabolism in mediating HCV assembly and egress, we examined Tg-mediated effects on lipid homeostasis. Tg treatment upregulated the expression of lipid synthesis genes, including FASN and DGAT1/2, and led to the accumulation of enlarged LDs. Tg also induced expression of CIDE-C, a mediator of LD fusion. Silencing CIDEC expression impaired Tg-induced LD enlargement and rescued viral RNA replication, but not extracellular titer, demonstrating that Tg-mediated LD remodeling contributes to replication defects without significantly affecting assembly or egress. Intracellular viral titers were unchanged in Tg-treated cells, indicating intact virion assembly but a defect in secretion. Consistently, Tg treatment reduced apolipoprotein B secretion, but not that of Gaussia luciferase, suggesting that Tg specifically disrupts the lipoprotein secretion pathway, which is required for efficient HCV egress. Together, our findings reveal that modulation of lipid homeostasis by Tg inhibits HCV RNA replication and egress by distinct mechanisms. This work has antiviral implications for other viruses that rely on lipid metabolism during infection.

microbiology↗

bioRxiv2025

Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

Broad spectrum antivirals are critical to respond rapidly to the threat posed by newly emerging RNA viruses. One potential candidate is the natural compound thapsigargin (Tg). Tg potently induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Recent studies have demonstrated that Tg has robust antiviral activity against several human coronaviruses (CoVs), including SARS-CoV-2, although the specific antiviral mechanism(s) have remained unclear. Here, we aimed to characterize the role of the UPR in the antiviral activity of Tg against HCoV-229E, a model common cold CoV. Consistent with previous findings, we show that a short 30-minute priming of A549 cells with Tg potently inhibits HCoV-229E infection. Time-of-addition assays showed that Tg is most effective when added up to 8 hours post-infection. Furthermore, Tg inhibits the accumulation of double-stranded RNA in infected cells, suggesting that Tg inhibits early stages of viral RNA replication. Using selective UPR pathway inhibitors to narrow down the role of these pathways in mediating the antiviral effect of Tg, we show that the inhibition of IRE1 or ATF6 does not impair the ability of Tg to inhibit HCoV-229E infection. The use of stable knockdown A549 cells in which IRE1, PERK, or ATF6 expression was silenced further revealed that the antiviral activity of Tg is not dependent on the expression of any of the three UPR sensors individually. However, HCoV-229E replication is inhibited in A549-shIRE1 cells, or in cells treated with the IRE1 inhibitor (KIRA6), suggesting that IRE1 activation may play a pro-viral role during HCoV-229E infection. Selective UPR pathway activators were used to further probe down the role of each pathway during HCoV-229E infection. Selective activation of the PERK pathway, but not IRE1 or ATF6 pathways, inhibits HCoV-229E infection. Lastly, to more broadly test the antiviral role of PERK against CoV RNA replication, we used BHK-21 cells that stably express a SARS-CoV-2 replicon. We show that selective PERK activation robustly inhibits SARS-CoV-2 replication, comparable to Tg. Overall, these findings provide insight into the antiviral mechanism(s) of Tg against CoV infection and demonstrate that modulation of the UPR may be exploited as an antiviral strategy.

microbiology↗

bioRxiv2025

Deconstructing natural products to develop synthetic small molecule attachment inhibitors with broad spectrum antiviral activity

The continuous emergence of new viruses and the number of viruses that are each highly consequential for few people raise a need for broad spectrum antivirals. Most human and emerging viruses first attach to cellular glycosaminoglycans (GAG) or sialylated glycans (SG), a potential target for broad spectrum antivirals. Attachment to the former is through polar interactions between the negatively charged glycans and positively charged domains in virion proteins and typically inhibited by negatively charged polymers. Attachment to the latter is through specific interactions at binding pockets and inhibited by molecules that bind to these pockets. Surprisingly, EGCG inhibits viruses that attach to GAG and SG. However, it does so with widely differing potencies, is not a pharmacologically desirable molecule, and is limited by solubility. We tested whether it was possible to develop small synthetic molecules to inhibit viruses that attach to SG or GAG. We first identified the EGCG moieties responsible for the antiviral activity. The two polyhydroxylated phenyl groups were essential while the central benzopyran linker was not. We thus designed a series of gallate compounds to explore the minimal pharmacophore required for broad-spectrum antiviral activity. By exploring the linkers and number of galloyls, we identified small molecule inhibitors of herpes simplex virus 1, influenza A virus, and the coronaviruses hCoV OC43 and SARS-CoV-2. These compounds have low micromolar to submicromolar potency and no limiting cytotoxicity. These molecules are still not pharmacologically optimized and limited by solubility, but they define a minimal pharmacophore that confers broad spectrum antiviral activity. ImportanceThe SARS-CoV-2 pandemic demonstrated the importance of antivirals in managing emerging viruses. Although vaccines were successfully developed in less than two years, there was resistance to vaccination while infected or sick people were far more willing to take antivirals. It is impossible to develop antivirals specific for unknown viruses, but broad spectrum antivirals could control viral spread until more specific and potent drugs are developed. Human pathogenic and emerging viruses commonly attach to glycans, providing a target for broad spectrum antivirals. However, inhibitors of attachment to glycosaminoglycans do not typically inhibit viruses attaching to sialylated glycans, and vice-versa. We had found that EGCG has the unique property of inhibiting viruses that attach both glycans, with quite different potencies. Here, deconstructed a natural compound, EGCG, to identify the moieties responsible for its antiviral activity to then produce broad spectrum antiviral compounds against established and emerging viruses that attach to either glycan.

microbiology↗

bioRxiv2024

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown. In this study, we used genetic and enzymatic approaches to deplete sialic acid from cell surfaces and compared the requirement for sialoglycans during endosomal and cell-surface CoV entry, primarily using lentiviral particles pseudotyped with the spike proteins of different sarbecoviruses. We show that entry of SARS-CoV-1, WIV1-CoV and WIV16-CoV, like the SARS-CoV-2 omicron variant, depends on endosomal cathepsins and requires cellular sialoglycans for entry. Ancestral SARS-CoV-2 and the delta variant can use either pathway for entry, but only require sialic acid for endosomal entry in cells lacking TMPRSS2. Binding of SARS-CoV-2 spike protein to cells did not require sialic acid, nor was sialic acid required for SARS-CoV-2 entry in TMRPSS2-expressing cells. These findings suggest that cellular sialoglycans are not strictly required for SARS-CoV-2 attachment, receptor binding or fusion, but rather promote endocytic entry of SARS-CoV-2 and related sarbecoviruses. In contrast, the requirement for sialic acid during entry of MERS-CoV pseudoparticles and authentic HCoV-OC43 was not affected by TMPRSS2 expression, consistent with a described role for sialic acid in merbecovirus and embecovirus cell attachment. Overall, these findings clarify the role of sialoglycans in SARS-CoV-2 entry and suggest that cellular sialoglycans mediate endosomal, but not cell-surface, SARS-CoV-2 entry. Thus, it may be important to consider both cell entry pathways when developing sarbecovirus entry inhibitors targeting virus-sialoglycan interactions. Author summaryThe COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 676 million infections and 6.8 million deaths so far, demonstrating the threat posed by emerging CoVs. In humans, SARS-CoV-2 and related coronaviruses cause respiratory tract infections, such as the common cold, as well as more severe disease in some individuals. To prepare for future outbreaks, conserved steps in the CoV replication could be considered for antiviral prophylactic or therapeutic approaches. One such process is CoV cell entry, which occurs via two main routes: At the cell surface or within endosomes. Cellular receptors, proteases and complex sugars, known as glycans, mediate CoV entry steps. In this study, we compared the role of a specific glycan subset, sialoglycans, in endosomal and cell surface CoV entry. We show that sialoglycans are required for entry of various CoVs that are mainly dependent on the endosomal route, but in the case of SARS-CoV-2, sialoglycans were not required when the cell-surface entry route was available. Our findings contribute to understanding the mechanisms of CoV entry, which could inform development of pan-CoV antivirals that target CoV entry steps.

microbiology↗

bioRxiv2023

One-Step Selective Labeling of Native Cell-Surface Sialoglycans by Exogenous α2,8-Sialylation

Exo-enzymatic glycan labeling strategies have emerged as versatile tools for efficient and selective installation of glycan terminal motifs onto live cell-surfaces. Through employing specific enzymes and nucleotide sugar probes, cells can be equipped with defined glyco-epitopes for modulating cell function or selective visualization and enrichment of glycoconjugates. Here, we identify Campylobacter jejuni sialyltransferase Cst-II I53S as a tool for cell-surface glycan modification, expanding the exo-enzymatic labeling toolkit to include installation of 2,8-disialyl epitopes. Labeling with Cst-II was achieved with biotin- and azide-tagged CMP-Neu5Ac derivatives on a model glycoprotein and on native sialylated cell-surface glycans across a panel of cell lines. The introduction of modified Neu5Ac derivatives onto cells by Cst-II was also retained on the surface for 6 h. By examining the specificity of Cst-II on cell surfaces, it was revealed that the 2,8-sialyltransferase primarily labeled N-glycans, with O-glycans labeled to a lesser extent, and there was an apparent preference for 2,3-linked sialosides. This approach thus broadens the scope of tools for selective exo-enzymatic labeling of native sialylated glycans and is highly amenable for construction of cell-based arrays.

biochemistry↗

bioRxiv2023

Induction of antiviral gene expression by cyclosporine A, but not inhibition of cyclophilin A or B, contributes to its restriction of human coronavirus 229E infection in a lung epithelial cell line

The development of antivirals with an extended spectrum of activity is an attractive possibility to protect against future emerging coronaviruses (CoVs). Cyclosporine A (CsA), a clinically approved immunosuppressive drug, has established antiviral activity against diverse unrelated viruses, including several CoVs. However, its antiviral mechanisms of action against CoV infection have remained elusive, precluding the rational design of non-immunosuppressive derivatives with improved antiviral activities. In this study, we evaluated the mechanisms of CsA against HCoV-229E infection in a human lung epithelial cell line. We demonstrate that the antiviral activity of CsA against HCoV-229E is independent of classical CsA target proteins, cyclophilin A or B, which are not required host factors for HCoV-229E in A549 cells. Instead, CsA treatment induces expression of antiviral genes in a manner dependent on interferon regulatory factor 1, but independent of classical interferon responses, which contributes to its inhibitory effect against HCoV-229E infection. Our results also point to a role for the HCoV-229E nucleoprotein in antagonizing activation of type I interferon, but we show that CsA treatment does not affect evasion of innate immune signaling pathways by HCoV-229E. Overall, our findings further the understanding of the antiviral mechanisms of CsA against CoV infection and highlight a novel immunomodulatory strategy to inhibit CoV infection that may inform future drug development efforts.

microbiology↗

bioRxiv2021

The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus entry inhibitor

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective antiviral countermeasures are urgently needed as vaccine development and deployment against an emerging coronavirus takes time, even in the best-case scenario. The green tea catechin, epigallocatechin gallate (EGCG), has broad spectrum antiviral activity. We demonstrate here that EGCG prevents murine and human coronavirus infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from highly pathogenic coronaviruses, as well as a bat coronavirus poised to emerge into humans. We show that EGCG treatment reduces coronavirus attachment to target cell surfaces. Our results demonstrate the potential for the development of pan-coronavirus attachment inhibitors to protect against current and future emerging coronaviruses.

microbiology↗