Biology subjects

Pellizzari-Delano, I. E.

Publications and source records attributed to Pellizzari-Delano, I. E..

3 recordsLinked to original sources

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↗

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↗