Function and interaction of coronavirus ion channel proteins

Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.

Coronaviruses

The present chapter is based on a text by John Ziebuhr published previously as a book chapter (in German) in "Medizinische Mikrobiologie und Infektiologie" (9th ed., S. Suerbaum et al. [eds.], Springer, 2020). © Springer-Verlag GmbH Germany, part of Springer Nature 2022. All rights reserved.

Therapeutic Effects of Renin-Angiotensin Systeminhibiting Drugs Losartan and Enalapril on Sars-Cov-2 Infection in Ex Vivo Cultured Human Precisioncut Lung Slices

The role of the renin-angiotensin system (RAS) in coronavirus disease 2019 (COVID-19) has received much attention, because the angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been speculated that, in COVID-19, RAS dysregulation in favor of angiotensin II (Ang-II)-mediated signaling might result in severe tissue inflammation and lung injury. Likewise, the role of a pre-existing therapy with angiotensin-converting enzyme 1 inhibitors (ACEi) or Ang- II type 1 receptor blockers (ARBs) in COVID-19 is largely unclear. We evaluated the effects of the ACEi enalapril (ENA) and the ARB losartan (LOS) on SARS-CoV-2 infection in human ex vivo-cultured, precision-cut lung slices (PCLS) obtained from normal human lung tissue. The PCLS were pretreated for 5 days with vehicle, LOS or ENA (300 muM), followed by mock infection or infection with SARS-CoV-2 and (continued) incubation with vehicle, LOS or ENA for 1 or 2 days. Thereafter, PCLS were harvested for analysis of viral replication, inflammatory responses, endoplasmic reticulum (ER) stress and apoptosis pathways. Both LOS and ENA significantly reduced viral replication in PCLS, with ENA being more potent. LOS was more efficient than ENA in reducing the expression of IL1B, CCL2, CXCL2 and TNFA, but not of IL6, whereas ENA preferentially caused a reduction of IL6 and CCL2 in SARS-CoV-2-infected PCLS. Furthermore, ENA, but not LOS, significantly decreased the expression of viral entry factors, ACE2 and transmembrane serine protease 2 (TMPRSS2), in infected PCLS, both of which were found to be robustly induced upon SARS-CoV-2 infection. Importantly, LOS or ENA did not exert apoptosis or other cytotoxic effects. Renin-angiotensin system-antagonizing drugs do not seem to exert detrimental effects during SARS-CoV-2 infection.On the contrary, in an ex vivo model of human PCLS, such treatment was found to dampen SARS-CoV-2 infection and consecutive inflammation.

Characterization of a putative human coronavirus (HCoV-229E)-encoded ion channel protein

Introduction: Acute respiratory tract infection is the major cause for morbidity in human coronavirus (CoV) infections including COVID-19. Approximately 30% of the patients hospitalized with COVID-19 also developed kidney injury. Although the molecular basis remains to be characterized, there is initial evidence to suggest that both indirect (hypoxia and hypotension, cytokine storm, etc.) and direct effects (infection via ACE2 present in the apical border of the proximal tubular cells and in podocytes) contribute to this kidney injury. A detailed understanding of the molecular biology and pathogenesis of coronaviruses is essential for providing better patient care and for facilitating the development of therapeutics. In this study, we sought to characterize a putative ion channel function of an accessory protein (ap) that is conserved in alphacoronaviruses and referred to as ap4 in human coronavirus 229E (HCoV-229E). We characterized the full-length ap4 protein encoded by clinical isolates of HCoV-229E and a C-terminally truncated version of this protein (ap4a) that is expressed by a cell culture-adapted strain of HCoV-229E. Proteins related to ap4 are also encoded in the genomes of other coronaviruses (between the S and E genes) and there is preliminary evidence that some of these proteins act as viroporins and have a role in viral replication and egress. Methods: Recombinant HCoV-229E strains with different forms of ap4 were propagated in Huh-7 cells, MRC-5 cells and human monocyte derived macrophages. Viral growth kinetics was determined and changes in the ap4 coding regions were studied by sequence analysis of virus progeny collected after serial passaging in these cell types. Also, a potassium uptake assay was performed in a Saccharomyces cerevisiae strain lacking the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) technology. Results: Serial passaging demonstrated the acquisition of frameshift mutations in ORF4 resulting in C-terminally truncated protein versions, indicating a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length accessory protein ap4 was expressed compared to the C-terminally truncated protein ap4a. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a nonselective channel or weak K+ channel with the following selectivity filter range: K+<Na+, Rb+<Cs+. Discussion: Our study provides the first evidence that ap4 acts as a viroporin. Further studies are warranted to (i) determine the precise role of ap4 in viral replication by modulating host cell ion homeostasis and (ii) explore if ap4 represents a potential antiviral drug target.