The current pandemic of coronavirus disease 2019 (COVID-19) has spurred intense interest into the mechanisms of cell damage and tissue death following infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
A new preprint, available on the bioRxiv* server, describes the varying ways in which the virus co-opts the machinery of the host cell following infection to evade or modulate the host immune response, alter the pattern of translation of viral proteins, and the release of new viral particles.
Hijacking the host cell
Among the most important changes that result from SARS-CoV-2 infection are the use of protein synthetic organelles and pathways to produce viral proteins and their post-translational modification by breaking up proteins, creating new disulfide bridges, adding phosphate groups, and especially, glycosylation.
The importance of glycosylation
Glycosylation refers to the trafficking of newly synthesized viral proteins through the endoplasmic reticulum (ER) and Golgi apparatus, where glycans are added to them, especially N-linked glycans. The level of glycosylation and protein folding is regulated within these organelles.
This type of processing is often used to escape the host immune response by preventing virus recognition by the host; to enhance virus-receptor binding; increase viral infectivity and release from the cell; and increase virulence as well as promote virion release. This makes it an obvious and important therapeutic antiviral target.
For instance, if glycoprotein folding is disrupted by inhibiting N-linked glycosylation, or simply if the level of glycosylation is directly altered, SARS-CoV-2 infection could be targeted.
Secondly, unregulated viral protein synthesis by the hijacked cell machinery may rapidly exceed the capacity of the ER to fold proteins properly. The high level of unfolded proteins induces ER stress, which in turn triggers compensatory pathways called the unfolded protein response (UPR).
Viral proteins are glycosylated, particularly the SARS-CoV-2 spike protein at up to 35% of its composition. This affects the infectivity and susceptibility to immune neutralization. ER and Golgi compartment processing allows the cell to inhibit glycosylation, and perhaps suppress maturation, thus regulating virion assembly.
The SARS-CoV-2 ORF8 (open reading frame 8) protein of the virus also interacts with several ER proteins, interfering with interferon-I release and thereby hampering antiviral immunity.
Unfolded protein response
The result of UPR is negative feedback on protein synthesis, enhanced ER folding capacity, and diversion of misfolded proteins to be broken down within proteasomes. It comprises three signaling pathways initiated by the three protein sensors IRE1, PERK and ATF6.
IRE1 activation results in the upregulation of genes involved in the ER stress response, while PERK activation results in phosphorylation. This, in turn, activates other transcriptional factors that lead to ATF4 expression at higher levels. The result is to reduce protein synthesis while inducing some other UPR-related transcription factors.
ATF6 release from the ER membrane is followed by cleavage within the Golgi apparatus. The activated form enhances the expression of ER chaperone-encoding genes and other genes required for the breakdown of the unfolded proteins.
Combining analysis of the complete protein profile of the infected host cell with that of microsomal proteins and deglycosylated proteins, the researchers found that indeed, SARS-CoV-2 induces stress on the ER leading to a UPR along with a reduction in glycosylation within the host cell.
These responses were already occurring at 2 hours post-infection (hpi), indicating that the host glycoproteins were undergoing remodeling. The majority of proteins in the host proteome were downregulated at 48 hpi, while viral proteins increased dramatically in abundance, reflecting viral replication.
Apoptosis of infected cells via UPR
Conversely, apoptosis-related proteins such as cleaved caspase-3 were higher, with increased rates of protein folding, at this point or even earlier. Oxidative stress markers were also increased, despite an early rise in anti-oxidant activity in the infected cell. All cell lines did not show equal rates of apoptosis, however, indicating that they enter these cell signaling pathways at different rates. This could be because the host cell simultaneously activates enhanced homeostatic responses to remain viable.
The UPR was also confirmed to be due to viral infection, mediated by ER stress in these cells. Apoptosis is due to this pathway, as well as by NRF2-mediated signaling.
Thus, infection reduced protein synthesis at all time points, except for an early increase at 6 hpi.
What are the implications?
These findings reflect those of earlier studies indicating that virus-induced activation of the UPR pathways enhances the expression of ER chaperones, thus facilitating infection. ER stress activates apoptosis pathways and may also cause increased oxidative stress by reactive oxygen species, mediated by pro-oxidant cytokines such as tumor necrosis factor (TNF). The eventual outcome is cell death at a later stage of infection, at 48 hpi.
Patients with severe complicated COVID-19 show ER stress markers such as phosphorylated PERK in their lung tissue. This also occurs in kidney and liver cells. The higher the ER transcriptional level, the longer the survival of the patient.
The key role played by ER stress and UPR indicate their importance as a therapeutic approach for the COVID-19 pandemic.
These data highlight the importance of ER-stress and UPR modulation as a host regulatory mechanism during viral infection and could point to novel therapeutic targets,” conclude the researchers.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.