Electrophysiological Impact of SARS-CoV-2 Envelope Protein in U251 Human Glioblastoma Cells: Possible Implications in Gliomagenesis?

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, the acute respiratory disease which, so far, has led to over 7 million deaths. There are several symptoms associated with SARS-CoV-2 infections which include neurological and psychiatric disorders, at least in the case of pre-Omicron variants. SARS-CoV-2 infection can also promote the onset of glioblastoma in patients without prior malignancies. In this study, we focused on the Envelope protein codified by the virus genome, which acts as viroporin and that is reported to be central for virus propagation. In particular, we characterized the electrophysiological profile of E-protein transfected U251 and HEK293 cells through the patch-clamp technique and FURA-2 measurements. Specifically, we observed an increase in the voltage-dependent (Kv) and calcium-dependent (KCa) potassium currents in HEK293 and U251 cell lines, respectively. Interestingly, in both cellular models, we observed a depolarization of the mitochondrial membrane potential in accordance with an alteration of U251 cell growth. We, therefore, investigated the transcriptional effect of E protein on the signaling pathways and found several gene alterations associated with apoptosis, cytokines and WNT pathways. The electrophysiological and transcriptional changes observed after E protein expression could explain the impact of SARS-CoV-2 infection on gliomagenesis.

Measuring infectious SARS-CoV-2 in clinical samples reveals a higher viral titer:RNA ratio for Delta and Epsilon vs. Alpha variants.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein.

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines.

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for ß-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of ß-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.