SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives.

Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs), and the DNAJ chaperones. This review will focus on the pleiotropic roles of ER chaperones during viral infection. We will cover their essential role in the folding and quality control of viral proteins, notably viral glycoproteins which play a major role in host cell infection. We will also describe how viruses co-opt ER chaperones at various steps of their infectious cycle but also in order to evade immune responses and avoid apoptosis. Finally, we will discuss the different molecules targeting these chaperones and the perspectives in the development of broad-spectrum antiviral drugs.

Novel SARS-CoV-2 variants induce higher toxicity in cardiovascular cells

Objective: SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19) and has spawned a global health crisis. Virus infection can lead to elevated markers of cardiac injury and inflammation associated with a higher risk of mortality. However, it is so far unclear whether cardiovascular damage is caused by direct virus infection or is mainly secondary due to inflammation. Recently, additional novel SARS-CoV-2 variants have emerged accounting for more than 70% of all cases in Germany. To what extend these variants differ from the original strain in their pathology remains to be elucidated. Here, we investigated the effect of the novel SARS-CoV-2 variants on cardiovascular cells. Results: To study whether cardiovascular cells are permissive for SARSCoV-2, we inoculated human iPS-derived cardiomyocytes and endothelial cells from five different origins, including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells, in vitro with SARS-CoV-2 isolates (G614 (original strain), B.1.1.7 (British variant), B.1.351 (South African variant) and P.1 (Brazilian variant)). While the original virus strain infected iPS-cardiomyocytes and induced cell toxicity 96h post infection (290±10 cells vs. 130±10 cells;p=0.00045), preliminary data suggest a more severe infection by the novel variants. To what extend the response to the novel variants differ from the original strain is currently investigated by phosphoproteom analysis. Of the five endothelial cells studied, only human coronary artery EC took up the original virus strain, without showing viral replication and cell toxicity. Spike protein was only detected in the perinuclear region and was co-localized with calnexin-positive endosomes, which was accompanied by elevated ER-stress marker genes, such as EDEM1 (1.5±0.2-fold change;p=0.04). Infection with the novel SARS-CoV-2 variants resulted in significant higher levels of viral spike compared to the current strain. Surprisingly, viral up-take was also seen in other endothelial cell types (e.g. HUVEC). Although no viral replication was observed (850±158 viral RNA copies at day 0 vs. 197±43 viral RNA copies at day 3;p=0.01), the British SARS-CoV-2 variant B.1.1.7 reduced endothelial cell numbers (0.63±0.03-fold change;p=0.0001). Conclusion: Endothelial cells and cardiomyocytes showed a distinct response to SARS-CoV-2. Whereas cardiomyocytes were permissively infected, endothelial cells took up the virus, but were resistant to viral replication. However, both cell types showed signs of increased toxicity induced by the British SARS-CoV-2 variant. These data suggest that cardiac complications observed in COVID-19 patients might at least in part be based on direct infection of cardiovascular cells. The more severe cytotoxic effects of the novel variants implicate that patients infected with the new variants should be even more closely monitored.

A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding.

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.