Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF3a protein plays a vital role in viral pathogenesis and coronavirus disease 2019 (COVID-19). Like the spike protein, ORF3a mutates frequently, and certain variants are associated with the severity of COVID-19. Given the clinical significance and functional implications of ORF3a mutations, we conducted a comprehensive mutagenesis study targeting various known functional elements and revealed two distinctive types of ORF3a proteins based on their subcellular localizations: ORF3a proteins primarily localize on the lysosomal membrane (L-ORF3a) and those present in the endoplasmic reticulum (E-ORF3a). The objective of this study was to contrast the functional and mechanistic distinctions between these two types of ORF3a proteins. We examined six distinct ORF3a mutants and assessed their effects on cellular oxidative stress, nuclear factor kappa B-induced cytokine production, and cell death. Mechanistically, we explored ORF3a-induced ER stress, autophagy, and interactions with relevant cellular proteins. Our findings indicate that ORF3a proteins induce cytopathic effects through a similar mechanism, irrespective of their subcellular location. However, E-ORF3a proteins elicit more pronounced cytopathic effects despite their lower abundance and minimal impact on ER stress and autophagy when compared to L-ORF3a proteins. This discrepancy is attributed to ER-associated degradation since ORF3a proteins bind to a ubiquitin E3 ligase TRIM59. Inhibition of the 26S proteasome partially restores the protein levels of E-ORF3a and cellular ER stress response. This suggests that even a small quantity of ORF3a can lead to significant cytopathic effects due to the delicate nature of ER. Our study underscores the intricate interplay of dynamic cellular signaling within these two subcellular compartments in response to ORF3a. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

Insights into the Activation of Unfolded Protein Response Mechanism during Coronavirus Infection

“…For instance, lysosomal ORF3as can induce the IRE1-XBP1s pathway of the UPR and autophagy more efficiently compared to ER-residing ORF3as. However, the ER-ORF3as, although they are eventually degraded by the ERAD mechanism, demonstrate more intense cytopathic responses via the ER stress-reticulophagy response [110]. Moreover, there are suggestions that some of the other accessory proteins can interact with the UPR mechanism too.…”

SARS-CoV-2 ORF3a Protein as a Therapeutic Target against COVID-19 and Long-Term Post-Infection Effects

“…Consequently, ORF3a plays an important role in the formation of ER-derived compartments such as DMVs that are associated with viral replication and transcription in host cells [46]. After protein synthesis of SARS-CoV-1 and SARS-CoV-2 ORF3a in the ER (32), ORF3a is transported from the ER to the Golgi apparatus, where it undergoes posttranslational modification via O-glycosylation before insertion into the plasma membrane and endomembranes, including endosomes and lysosomes [31,[47][48][49]. This delineates a dynamic intracellular movement process for the ORF3a protein following its production.…”

“…This delineates a dynamic intracellular movement process for the ORF3a protein following its production. Functional domains within ORF3a, such as the YXXΦ motif (aa160-163) and the diG motif (aa187-188) in the cytoplasmic region, drive the transport of ORF3a from the ER to plasma membranes and other endomembranes [31,33,34,47].…”

Effect of Melatonin on Herpesvirus Type 1 Replication

“…Thus, the new viral demand for proteins will induce an excess of endoplasmic request that will result in an overproduction of misfolded proteins known as endoplasmic reticulum stress [4], which usually exceeds the capacity of the proteasome. To try to counteract this stress, the cell develops its multifactorial misfolded protein response system (known as UPR) which triggers consecutive response cascades depending on the severity of the induced stress [5] and which may ultimately induce the activation of survival mechanisms, such as autophagy or programmed cell death or apoptosis. Melatonin (N-acetyl-5methoxytryptamine) is a highly conserved multifunctional signalling molecule.…”