SARS-CoV-2 hijacks antiviral factors and promotes human lung cell infection

The virus behind the current COVID-19 pandemic, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is known to spread more efficiently than previous pathogenic viruses Coronavirus, SARS-CoV, and MERS-CoV. However, so far the mortality rate has been much lower at 2% to 5%, 10% for SARS and about 40% for MERS. Scientists believe that the virus will be blocked by interferon (IFN) more than previous viruses. In fact, IFN is currently used to reduce the severity of COVID-19.

New research now published on preprint server bioRxiv* Under conditions similar to them in August 2020 In vivoInstead, IFN may facilitate efficient virus entry.

This scanning electron microscopy image shows SARS-CoV-2 (a magenta round object) emerging from the surface of cells cultured in the laboratory. SARS-CoV-2 (also known as 2019-nCoV) is a virus that causes COVID-19. The virus shown was isolated from a patient from a colored US image captured at NIAID’s Rocky Mountain Laboratories (RML) in Hamilton, MT. Credit: NIAID

What is IFITM?

Interferon-induced transmembrane proteins (IFITM 1, 2, and 3) are proteins that are believed to inhibit various viruses, including SARS-CoV. Most of the evidence for this comes from studies using cells that overexpress these proteins and are infected with pseudoviruses.

The researchers looked for innate immune effectors that target SARS-CoV-2 entry into target cells. Viral invasion involves spike-mediated recognition of the host receptor angiotensin converting enzyme (ACE) 2 and causes irreversible structural changes. Spike protein In its fused form by proteolytic cleavage into S1 and S2 subunits. The cleaved protein fuses with the plasma membrane and enters the cell.

IFITM is a family of IFN-stimulated genes (ISG), which is known to prevent this fusion in influenza A virus, rhabdovirus, and HIV.

IFITM overexpression inhibits pseudoviral S binding

Previous studies have shown that when these proteins are expressed at excessively high levels, pseudoparticles expressing the SARS and MERS spike proteins are unable to enter the host cell. The mechanism of inhibition may reduce the rigidity and curvature of the plasma membrane such that fusion cannot occur. IFITM1 is at the plasma membrane only, whereas IFITM2 and 3 are localized to intracellular lysosomal membranes. Many scientists believe that such viruses are unable to replicate in cells expressing these proteins.

However, some studies have shown that IFITM may actually increase the intensity of infection with some human coronaviruses. At the same time, the mutated IFITM may enhance infection by many viruses of this family, including SARS.

Current studies show that overexpression of IFITM is reduced by two orders of magnitude, especially in IFITM2 and IFITM3, reducing pseudoparticle invasion via the SARF-CoV-2 spike, and less intense in IFITM1. The infectivity of these pseudoviruses did not diminish, but in fact the spike proteins Fake virus..

Initial tests showed that both pseudovirus-expressed SARS-CoV and SARS-CoV-2 spike proteins were efficiently inhibited by IFITM. The mechanism of such inhibition seems to be through ubiquitination and palmitoylation.

In all cases, they found that IFITM reduced cell-cell fusion mediated by spiking ACE2 binding. Depletion of these proteins resulted in a 3- to 7-fold increase in spike-mediated infection by all pseudoviral particles. Further testing with cell lines lacking IFITM expression jumped the number of S-ACE2 binding foci from 4-fold to 10-fold. These findings strongly suggest that the IFITM protein is an efficient inhibitor of SARS-CoV-2S-mediated viral entry.

The question is whether this susceptibility to inhibition of S-ACE2 binding by IFITM is shared by SARS-CoV-2 even after transfecting human hosts across species barriers.

Figure S1 (related to Figure 1). VSV-G mediated infection by VSVpp is not significantly inhibited by the IFITM protein. (A) VSV (luc) due to luciferase activity in HEK293T cells transiently expressing the indicated proteins and infected with VSV (luc) ΔG* VSV-G (MOI 0.025) for 16 hours 24 hours after transfection. Quantification of ΔG*VSV-G entries. Bars represent the average of n = 3 ± SEM. Lower panel: Immunoblot of corresponding whole cell lysates (WCL) stained with anti-IFITM1, anti-IFITM2, anti-IFITM3, anti-PSGL-1, anti-ACE2 and anti-actin. (B) Immunoblot analysis of whole cell or supernatant lysates of HEK293T cells transiently transfected with increasing doses of SARS-CoV-2 S VSVpp and IFITM1 expression constructs. Blots were stained with anti-spikes, anti-VSV-M, anti-IFITM1 and anti-actin. (C) VSV (luc) ΔG* VSV-G particles due to luciferase activity in Caco-2 cells infected with supernatant of HEK293T cells transiently transfected with VSV-G and empty control or IFITM expression vector Quantitative. Bars represent the average of n = 3 ± SEM. (D) Exemplary immunoassays of whole cell lysates of Calu-3 cells transiently transfected with control siRNA (si.NT) or siRNA targeting IFITM1, 2 or 3 as shown. Blot. Percentages indicate the signal strength of the three IFITM proteins compared to that observed in the presence of control siRNA (100%).

IFITM reduces virus load

Scientists have discovered that in response to interferon, IFITM is produced in the lung, the main site of infection by SARS-CoV-2. It also triggers on other infected tissues. IFN has both positive and detrimental effects on tissues and can exacerbate the disease and cause severe COVID-19. They also activate a cascade of cytokines and chemokines for inflammation, with an as yet unknown effect on disease.

The experiment was repeated using authentic SARS-CoV-2 in cell lines expressing the ACE2 receptor alone or with IFITM. They found that overexpression of IFITM1 or 2 dramatically reduced viral RNA loading by more than 30-fold and 136-fold. However, IFITM3 resulted in a 5-fold inhibition.

Endogenous IFITM enhances SARS-CoV-2 infection of Calu-3 cells

IFITM2 and 3 are constitutively expressed at low levels in primary human lung bronchial epithelium and Calu-3 cells that express both ACE2 and all IFITMs. Expression of all IFITMs was enhanced by treatment with IFN, but reached different levels depending on cell type. IFN-β was active in intestinal organoids, but IFN-β and IFN-γ were most efficient at increasing IFITM1 and IFITM2 in bronchial epithelial cells and IFITM1 in Calu-3 cells.

The researchers tested the effect of IFITM on the Calu-3 cell line. They found that when the wild-type virus was used to infect the human lung cell line, Calu-3, endogenous IFITM expression enhanced the infection with and without interferon. The most important IFITM in this process is IFITM2, which increased viral entry and production by several orders of magnitude. However, IFITMS was unable to potentiate SARS-CoV-2 infection of HEK cells with different levels of expression.

When these endogenous proteins were suppressed by siRNA knockdown, authentic SARS-CoV-2 replication fell 3-4-fold in IFITM1 and IFITM3 and 22-fold in the absence of IFITM2. After 24 hours, IFITM2 reduced virus load by more than two orders of magnitude.

Therefore, the endogenous expression of IFITM is not restricted and increases the entry and replication of this virus in human lung cells. Treatment with IFN-β resulted in a 22-fold reduction in viral RNA levels 48 hours after infection, and a further 4- to 68-fold reduction in silencing IFITM.

Infectious titer was examined by Vero cell cytopathic effect (CPE) assay. This confirmed that silencing all IFITM significantly reduced the infectivity. When only IFITM1 or IFITM3 were depleted, CPE was conservative and occurred only at low dilutions. Depletion of IFITM2 abolished CPE, and the lack of this protein was shown to reduce infectious viral particle yields by more than four orders of magnitude.

Implications and future direction

They concluded that IFN enhanced the baseline expression of IFITM protein in lung cells and other tissues infected with SARS-CoV-2, allowing the virus to spread in vivo. Therefore, Calu-3 cells are an excellent model for studying this infection.

In other words, the researchers say, “The IFITM expression is important for efficient SARS-CoV-2 replication in Calu-3 cells. [The virus uses this protein] To achieve efficient replication and diffusion under physiological conditions. “ It may also contribute to severe COVID-19 as antiviral factors allow the virus to efficiently infect cells in the lower respiratory tract.

This study also shows how important it is to confirm the observation of antiviral effects under in vivo conditions. Earlier investigators may have missed the enhancing effect because it is specific to the endogenously affected IFITM in human lung cells infected with wild-type virus. Antibodies to these proteins could be an interesting new treatment for this infection.

*Important Notices

bioRxiv It publishes preliminary, non-peer reviewed scientific reports and should not be considered conclusive and should not guide clinical practice/health-related behavior or be treated as established information.