SARS-CoV-2 hijacks lung cells for its own purposes

By Samantha Black, PhD, ScienceBoard editor in chief

December 3, 2020 -- The first map of molecular responses of human lung cells to SARS-CoV-2 infection has been developed. It shows that the novel coronavirus causes significant protein and phosphorylation damage, effectively hijacking lung cells to enable its entry and propagation, according to a study published in Molecular Cell.

In the distal lung, SARS-CoV-2 preferentially infects alveolar epithelial type 2 cells (AT2s) which express the angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease, serine 2 (TMPRSS2). These cells are progenitors of lung alveoli. Most COVID-19 mortality and morbidity results from alveolar injury, and therefore identifying mechanisms of how SARS-CoV-2 drives lung pathology in these cells is an urgent unmet need.

Alveolar epithelial type 2 cells derived from human-induced pluripotent stem cells (abbreviated as iAT2s) can be cultured to reveal mechanisms of SARS-CoV-2 propagation. Previous research in Vero E6 cells has shown that the earliest stage of infection occurs within one hour after exposure. Throughout the next eight hours, viral replication-transcription complexes accumulate to complete replication.

A collaborative group of researchers from the National Emerging Infectious Disease Laboratories (NEIDL), the Center for Regenerative Medicine (CReM), and the Center for Network Systems Biology at Boston University School of Medicine analyzed bioengineered human alveolar cells with tandem mass spectrometry to discern novel proteins and molecular pathways of SARS-CoV-2 in lung cells.

The researchers performed a deep quantitative temporal phospho/proteomic analysis to quantify cytopathologic changes induced by SARS-CoV-2 infection in iAT2s at four time points (one, three, six, and 24 hours post infection), with a focus on early events following viral entry. Total protein from culture was extracted and analyzed by precision mass spectrometry to quantify changes in the proteome and phosphoproteome relative to controls.

In total, they quantified 8,471 proteins including eight viral proteins, with 14,289 phosphosites on SARS-CoV-2 and 2,703 host phosphoproteins. Overall, they observed changes in 2,872 proteins. They identified 4,688 differential phosphosites mapping onto 1,166 unique lung proteins across all time points, many of which corresponded to regulators of pulmonary cell function. From these results, the researchers conducted in-depth differential pathway analysis and functional modeling.

By analyzing host responses to infection, the team identified four waves of host protein expression corresponding to immediate (one hour post infection), early (three hours post infection), and intermediate/late (three to six hours post infection) stages of viral infection. Infection over this period throws lung cells into disarray, causing abnormal changes in protein levels and frequency of phosphorylation (regulates protein function) inside cells. These changes help the virus multiply and eventually destroy cells, ultimately resulting in lung injury.

"The virus uses these resources to proliferate while evading attack by the body's immune system. In this way new viruses form which subsequently exit the exhausted and brutally damaged lung cell, leaving them to self-destruct. These new viruses then infect other cells, where the same cycle is repeated," explained corresponding author Andrew Emili, PhD, professor of biochemistry at BUSM, in a statement.

Specifically, the researchers found that during immediate infection, AT2 cells were enriched with factors tied to viral infection including markers of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated cytokine induction; activation of the protein complex, nuclear factor kappa B (NF-κB); and C-type lectin receptors.

Regulation of translation initiation by mammalian target of rapamycin (mTOR) was altered immediately following viral entry. This caused significant remodeling of host protein synthesis and altered levels of both the 40S and 60S ribosomal subunits during the later stages of infection. Furthermore, the mitochondrial 28S ribosomal components increased in late infection, likely to accommodate energetic demands for viral replication.

During the early to late stages of infection (three to 24 hours post infection) AT2 cells were enriched for proteins linked to cell proliferation, which included the dysregulation of the ataxia-telangiectasia mutated and RAD3-related protein (ATR; checkpoint kinase regulator), a caspase (CASP7/8; cell death effectors), and the mitotic checkpoint serine/threonine-protein kinase BUB1 beta (BUB1B; mitotic checkpoint regulator).

"Our results showed that in comparison to normal/uninfected lung cells, SARS-CoV-2 infected lung cells showed dramatic changes in the abundance of thousands of proteins and phosphorylation events," said Dr. Darrell Kotton, professor of pathology & laboratory medicine at BUSM and director of the CReM.

The data point to growth arrest and apoptosis in infected iAT2s, including hyperphosphorylation of the epidermal growth factor receptor (EGFR) at S991 (site linked to internalization), DNA damage response in infected iAT2s, hypoactive cell cycle kinases, and evidence of activated pro-apoptotic kinase CAMK2D. This indicates that SARS-CoV-2 disrupts multiple signaling pathways to cause cell death of AT2s.

"Moreover, our data also showed that the SARS-CoV-2 virus induces a significant number of these changes as early as one hour post infection and lays the foundation for a complete hijack of the host lung cells," adds Elke Mühlberger, PhD, associate professor of microbiology and principal investigator at the NEIDL.

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