How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1α through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.
In this study using the integrated proteo-transcriptomics studies we identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. The data therefore points towards dysregulation of mTOR/HIF-1 signaling cascades, which could be potential target for COVID-19 therapeutic interventions.
The mTOR signaling pathways are known to regulate apoptosis, cell survival, and host transcription and translation and can be hijacked by several RNA viruses like influenza virus and coronaviruses.PI3K activation results in AKT phosphorylation and subsequent activation of mTOR. Through a cascade of events, mTORC1 and AKT activates 4E-BP1 and eIF4 complex followed by translation of effector protein HIF-1α that initiates host transcription and translation of specific genes. Another pathway that changed over time was the TNF signaling pathway. TNF signaling is also interlinked with HIF-1 signaling and can induce HIF-1α through AKT and MAPK activation.Of note, specific proteins dysregulated in the TNF signaling pathway were caspase , caspase and CCAAT/enhancer-binding protein beta (CEBPB),which are linked to interferon (IFN) signaling and NF-κB signaling pathways. Previous studies on coronaviruses suggest a critical role of the IFN response, in particular IFN-β. This is also reflected in our findings that SARS-CoV-2 infections result in significantly dysregulated RIG-I, NLR and NF-κB pathways, which needs further evaluation. All these pathways have been linked to the IFN response.
It has been shown that absence of HIF-1α can promote replication of influenza A virus and severe inflammation mediated via promotion of autophagy. There could be several mechanisms that result in decreased phosphorylation of mTOR effectors such as the host response affecting the translation machinery in response to stress or viral proteins at the late stage of infection that promote the translation of viral mRNAs by shutting down host mRNA translation. Nonetheless, similar to other viruses that hijacking the AKT/mTOR pathway such as the highly pathogenic influenza virus and the Middle East respiratory syndrome coronavirus (MERS-CoV),deregulation of the mTOR pathway might enable SARS-CoV-2 to enhance its pathogenicity
A recent drug target network analysis based on potential human coronavirus and host interactions predicted that sirolimus (also known as rapamycin), which targets mTOR, could be repurposed.Sirolimus was shown to inhibit MERS-CoV infection by 60% in mice. Some studies have shown that everolimus, another mTOR inhibitor, and sirolimus are weakly active against influenza A virus.Everolimus delayed death but was not able to reduce mortality in lethal mouse infection model of influenza A (H1N1 and H5N1). Sirolimus was even shown to negatively affect the lung pathology probably due to its immunosuppressive effect. It has also been reported to block viral protein expression and virion release, improving the prognosis in patients with severe H1N1 pneumonia and acute respiratory failure.On the other hand, rapamycin treatment was shown to degrade antiviral barriers and could thus be potentially harmful in pathogenic viral infections.In COVID-19 patients the severity of the disease is associated with a cytokine storm with markedly increased expression of interleukin 6 (IL-6) in the serum of severe cases.Interestingly, IL-6 can activate mTOR in a STAT3 dependent or independent manner.Whether our proposed drugs can be indeed repurposed for COVID-19 therapies now needs to be carefully tested in in vitro SARS-CoV-2 infection models and in in vivo COVID-19 disease models.
There are some limitations of our study. First, we only used the HuH7 cell line but the SARS-CoV-2 can be also cultured in Vero E6, Vero CCL81, or HEK-293T cells. Whereas SARS-CoV-2 exerts rapid cytopathic effects in Vero E6 cells (within 24hpi), viral replication is slower in HuH7 and HEK293T cells, allowing to study host-cellular responses for 3 days after viral challenge. Moreover, an earlier study used HuH7 cells to identify the transcriptomics signature of early cellular responses to SARS-CoV and HCoV-229E infections . However, the observed effects could be cell type-specific and thus, we are currently assessing the effect of SARS-CoV-2 infection in other cells lines. Moreover, SARS-CoV-2 has a propensity to mutate and our experiments were performed with only one virus strain isolated from a Swedish patient. Of note, our virus isolate has close sequence similarity to the initial strains circulating in Wuhan, China.
In conclusions, we observed marked alterations of mTOR/HIF-1 signaling at the proteo-transcriptomic levels in response to SARS-CoV-2 infections, though the exact mechanistic role of these changes remains to be elucidated. Targeting mTOR/HIF-1 signaling could be an attractive candidate as a potential therapy, alone or preferably combined with antivirals, for the management of COVID-19 patients. Moreover, mTOR inhibition could be used to reduce the cytokine storm syndrome in severe cases of COVID-19.
Reference & source information: https://www.biorxiv.org/
Read More on: