Some of the most exciting discoveries in biology begin with questions that seem far removed from the final answer.
When we first started studying CLCC1 (Chloride Channel CLIC Like 1), we were interested in a fundamental cell biology question: how do cells maintain the unique internal environment of the endoplasmic reticulum (ER), and why is this important for neuronal survival?
Our journey began in the field of neuroscience, not virology. Through years of studying ER ion homeostasis, we discovered that CLCC1 is an ER-localized chloride channel. In 2023, our work showed that loss of CLCC1 disrupts ER homeostasis and causes neuronal dysfunction in mice (https://doi.org/10.1038/s41422-023-00798-z). At that time, we viewed CLCC1 primarily as a component of the cellular machinery that maintains organelle stability.
We never imagined that this ER ion channel would later become connected to coronavirus biology.
The unexpected connection emerged during the COVID-19 pandemic. Several studies reported that the SARS-CoV-2 accessory protein ORF3a interacts with CLCC1, but the biological meaning of this interaction remained unknown. Was CLCC1 simply another protein encountered by a viral factor, or was the virus revealing something fundamental about how host cells regulate their organelles?
Because our laboratory had already developed tools to monitor ER homeostasis, we realized that CLCC1 provided a unique opportunity to address this question.
Our first challenge was to move beyond a simple interaction. Viruses interact with hundreds of host proteins, but only a fraction of these interactions have functional consequences. We wanted to know whether ORF3a could alter CLCC1 biology.
Using CLCC1-TurboID proximity labeling, we found that ORF3a expression increased the labeling of CLCC1 molecules, suggesting that ORF3a might reorganize CLCC1 into a closer molecular environment, potentially altering the functional state of the channel.
The most memorable moment of this project happened during confocal microscopy. While looking at cells expressing ORF3a, I observed striking CLCC1 puncta structures through the eyepiece. All ORF3a-expressing cells displayed this phenotype, and the change was so obvious that it immediately caught my attention.
However, one thing puzzled me: although ORF3a clearly induced CLCC1 puncta formation, many of these puncta did not co-localize with ORF3a itself. This dissociation made us pause and rethink what was really happening.
Given the previously established role of ORF3a at lysosomes, we began to wonder: could CLCC1 be acting as a counterforce? We hypothesized that CLCC1 overexpression might function to retain ORF3a at the ER, potentially counteracting its normal lysosomal trafficking.
This hypothesis shifted the trajectory of the project. We no longer viewed CLCC1 as just a passive binding partner of ORF3a. Instead, we started to see it as an active player—a host factor that the virus might be exploiting to remodel ER organization and homeostasis.
In retrospect, this observation provided a critical conceptual turn: CLCC1 was not merely an interacting protein captured by ORF3a. It was a functional component of the cellular pathway altered by the viral protein.
Another unexpected discovery came from our investigation of autophagy.
Previous studies had demonstrated that ORF3a inhibits autophagic flux, and these findings were highly reproducible. However, our experiments suggested that ORF3a may promote ER-phagy, a selective autophagy pathway responsible for removing portions of the ER.
At first, this seemed contradictory. Could ORF3a both induce ER-phagy and inhibit autophagy?
Rather than assuming one observation was incorrect, we considered another possibility: perhaps we were observing different layers of the same complex process. Autophagy is not a single event. It involves cargo selection, membrane remodeling, lysosomal delivery, and degradation. Different experimental approaches may reveal different stages of this pathway.
To explore this possibility, we developed an improved ER-phagy reporter using the pH sensitivity of pHluorin. This approach allowed us to capture ER-phagy events that were difficult to detect previously. Our results suggested that ORF3a-induced ER-phagy and ORF3a-mediated inhibition of autophagic degradation represent distinct biological events occurring in different contexts.
Viruses do not necessarily create completely new pathways. Instead, they often reveal hidden relationships between existing cellular processes. In this case, SARS-CoV-2 ORF3a acted as a biological perturbation that exposed an unexpected connection between ER ion homeostasis, organelle remodeling, and selective autophagy.
The peer-review process further strengthened our understanding of this work. Reviewers raised important questions about the relationship between ER-phagy and autophagy inhibition, the biological significance of the ORF3a-CLCC1 interaction, and why we selected neuronal models. Addressing these questions required additional experiments and deeper thinking, but ultimately helped us refine the central message of the study.
Many questions remain.
We still do not know exactly how ORF3a reorganizes CLCC1 at the molecular level or how this affects channel function. The structure of the ORF3a-CLCC1 complex within cellular membranes remains an exciting mystery. Advanced approaches such as cryo-electron tomography may eventually reveal how viral proteins reshape host organelle architecture.
Looking back, the most important discovery was not simply that SARS-CoV-2 ORF3a interacts with CLCC1. The deeper insight is that a viral protein can uncover a previously hidden layer of organelle regulation.
We began this journey by asking how an ER ion channel supports neuronal homeostasis. Unexpectedly, a viral protein led us to discover new principles of how cells organize their internal environments. Sometimes, pathogens do not only teach us how diseases occur—they also reveal fundamental biology that was waiting to be discovered.