An international team of researchers has found that angiotensin-converting enzyme 2 (ACE2) levels in lung cells vary over the day, affecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The virus entry and replication in the lung cells also have a rhythmic cycle, suggesting circadian processes can affect the virus's life cycle.
Circadian rhythms, the internal processes that control sleep-wake cycles in organisms, regulate how the organism responds to the environment. Recent studies have shown that that the circadian clock also regulates the susceptibility of organisms to virus infection. Flu infection in mice lacking circadian regulating factors led to higher viral replication and severe bronchitis, suggesting a role for circadian function in respiration.
Almost all cells have circadian signals that are controlled by translation and transcription feedback loops. The molecular clock has a 24-hour periodicity. The transcription factor BMAL1-CLOCK activates the transcription in target gene promoters. Gene products provide feedback to repress the transcriptional activity of the activator.
In a new study published on the bioRxiv* preprint server, researchers report that the circadian pathways affect SARS-CoV-2) life cycle.
ACE2 levels show a rhythmic cycle
The authors infected lung epithelial cells Calu-3 with SARS-CoV-2 at different circadian times. They found that the virus showed a rhythmic pattern in replication, with the replication rate changing over 24 hours.
To check if this cyclic replication occurs at the level of virus entry, the researchers infected the cells with lentiviral pseudoparticles expressing the SARS-CoV-2 spike protein. The viral entry into the cells also followed a cyclic pattern. The infection increased and decreased over a 24-hour cycle, with the infection depending on the spike protein's interaction and the receptor ACE2. In contrast, pseudoparticles with the vesicular stomatitis virus (VSV) infected the cells with a similar efficiency at all times and showed no cyclicity.
SARS-CoV-2 infection is circadian
This suggests circadian signaling pathways play a role in modulating the susceptibility of Calu-3 cells to SARS-CoV-2 infection.
When the researchers measured the expression of ACE2 and TMPRSS2, another enzyme mediating SARS-CoV-2 infection, they found that ACE2 levels varied over 24 hours. There was a trough at 6 hours and a peak at 18 hours, coinciding with the SARS-CoV-2 infection. TMPRSS2 levels were constant throughout.
Circadian regulation is believed to be under transcriptional control, but quantification of the ACE2 and TMPRSS2 transcripts showed no cyclic pattern in Calu-3 cells. However, they saw some circadian patterns of expression in the lung, liver, and intestine of mice models to study circadian rhythms.
When Bmal1, a major circadian transcription activator, was silenced, Calu-3 cells showed reduced ACE2 expression, but there was no effect on TMPRSS2. These Calu-3 cells also showed reducing infection by the SARS-CoV-2 pseudoparticle. There was no difference in the VSV infection.
The team next tested the effect of a synthetic agonist, S9009, which modulates circadian pathways by activating REV-ERB, a BMAL1 repressor, on SARS-CoV-2 infection. Treating Calu-3 cells with S9009 reduced BMAL1 activity, reduced ACE2 but had no effect on TMPRSS2 expression, and S9009 also reduced SARS-CoV-2 infection. Treating primary bronchial epithelial cells with SR9009 also reduced infection by the SARS-CoV-2 pseudoparticle.
Circadian processes affect the SARS-CoV-2 life cycle
Thus, the results show circadian regulation of ACE2 expression in lung cells, which significantly affects SARS-CoV-2 infection. This suggests post-transcriptional and post-translational mechanisms may play a role in regulating the circadian clock. MicroRNAs have been shown to modulate the circadian clock and regulate ACE2 expression, providing a possible mechanism.
In addition, bioinformatics analysis suggested about 30% of the SARS-CoV-2 host factors could be regulated by BMAL1/REV-ERB. Experimentally, they found a significant reduction of SARS-CoV-2 replication in BMAL1 silenced Calu-3 cells compared to original cells. Thus, circadian processes could play a role in influencing several steps of the SARS-CoV-2 life cycle, including entry and replication.
Thus, there could be a potential application of drugs that modulate the circadian clock, such as dexamethasone, which has been shown to reduce COVID-19 severity and synchronize circadian pathways. Several compounds that target clock proteins have been developed recently and they have been shown to inhibit hepatitis C virus and HIV replication.
Circadian clocks can also affect drug responses. Night shift workers are at an increased risk of developing chronic inflammatory diseases. Hence, identifying if shift workers are more susceptible to SARS-CoV-2 infection could help public policy measures.
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.