SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines

Abstract

Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.

Keywords: SARS-CoV-2; glioblastoma; hepatoma; liver cells; permissiveness.

Figure 1

Overexpression of angiotensin-converting enzyme 2 (ACE2) confers the permissiveness of A549 cells to SARS-CoV-2. Naïve A549 (a–c) or Huh7.5 (d–f) cells or cells transduced by lentiviruses encoding ACE2 alone or together with TMPRSS2 were infected with SARS-CoV-2 and harvested 4 days post-infection (d.p.i.). Levels of ACE2 (a,d), TMPRSS2 (b,e) mRNA or SARS-CoV-2 RNA (c,f) were assessed by reverse transcription with real-time polymerase chain reaction (RT-qPCR). The levels were normalized to the levels of mRNA of β-glucoronidase (GUS). The values represent the means ± standard deviation (S.D.). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

SARS-CoV-2 establishes productive infection in human hepatoma but not in non-transformed hepatocyte-like cells or hepatic stellate cells. Cells were seeded on standard (a,b) or coated (c,d) cultured plates (HepG2 cells), incubated with SARS-CoV-2 at a MOI of 0.1 for 2 h, and levels of the virus RNA in cells (a,c) or conditioned medium (b,d) were quantified by RT-qPCR 4 days later. (e,f,) Levels of ACE2 (e) or TMPRSS2 (f) mRNA in cells presented on panel (a). The values are the means ± standard deviation (S.D.). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

SARS-CoV-2 induces a decrease in the expression of hepatocyte-specific genes. HepaRGdiff cells were infected and incubated in the presence of SARS-CoV-2 for 2 h and expression of albumin (a), cytochromes (CYP) 3A4 (b) and 2C9 (c) was quantified by RT-qPCR 4 d.p.i. The levels were normalized to the levels of GUS mRNA. The values represent the means ± standard deviation (S.D.). * p < 0.05.

Figure 4

SARS-CoV-2 may establish a productive infection in human high-grade glioblastoma. Glioblastoma U87-MG, U251-MG, DBTRG-05MG, high-grade low-passage glioblastoma (GBM3821, GBM4114, GBM5522 and GBM6138), neuroblastoma (SH-SY5Y, Kelly or La-N-1) or primary embryonic astrocytes were infected with SARS-CoV-2, 2 h later the medium was replaced, and levels of the viral RNA were assessed by RT-qPCR in cells (a) or conditioned medium (b). (c) The conditioned medium from GBM4114, GBM5522 and GBM6138 after infection was used to treat Vero E6 cells, and SARS-CoV-2 RNA in media was monitored by RT-qPCR. (d,e) Levels of ACE2 and TMPRSS2 mRNAs were measured in glioblastoma and neuroblastoma cell lines. The values are the means ± standard deviation (S.D.). (f,g) Immunostaining of the GBM6138 cell line infected with SARS-CoV-2 using non-immune serum (f) and immune (g) serum.

Figure 5

The hyperpermissive GBM6138 cell line has an impaired type I interferon system. Glioblastoma U87-MG, U251-MG, DBTRG-05MG, GBM3821, GBM4114, GBM5522 and GBM6138 were infected with Sendai virus, preinactivated with ultraviolet, and 24 hpi expression of interferons α (a) and β (b) as well as Mx1 (c) gene was assessed by RT-qPCR in cells. The values are the means ± standard deviation (S.D.). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.