SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression

Abstract

Aims: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication.

Main methods: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated.

Key findings: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro.

Significance: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

Keywords: ACE2; Autophagy; COVID-19; Cell lines; Mitochondria; TMPRSS2.

Fig. 1

SARS-CoV-2 kinetics in different cell types. A. Viral load 2 h post-infection (hpi) with SARS-CoV-2 (n = 3/group). One-way ANOVA followed by Tukey's test, F(8,18) = 23.33. B. SARS-CoV-2 extracellular (B1) and intracellular (B2) viral loads in different cell lines over time (n = 3–4/group). *p < 0.05. NS: non-significant. Mixed-effects model (REML) followed by Tukey's test, F(8,27) = 8.96 (B1), F(8,87) = 32.9 (B2).

Fig. 2

Cell viability after SARS-CoV-2 infection. A. Cell viability assessed in the presence or absence of SARS-CoV-2 over time (n = 3). Two-way ANOVA, followed by Tukey's post-test. **p < 0.001 and *p < 0.05. F(3,6) = 32.86 (Vero-E6), 0.63 (HuH7), 19.4 (SH-SY5Y), 0.68 (Huvec), 0.82 (Caco-2), 297.8 (A549), 5.48 (MCF7), 5.83 (HEK-293 T), 3.59 (BEAS-2B). B. Representative images were taken 24 h and 96 h in the presence (red) or absence (green) of SARS-CoV-2. Mock (non-infected cells). Scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Differential ACE2 and TMPRSS2 expression. A1. ACE2 expression according to Western blot analyses. F(8,18) = 21.21. A2. Histograms reporting the mean ± SD of ACE2 levels after normalization with the average intensity of the bands from three independent experiments. B1. TMPRSS2 expression according to Western blot analyses. F(8,18) = 20. B2. Histograms reporting the mean ± SD of TMPRSS2 levels after normalization with the average intensity of the bands from three independent experiments. C. ACE2 mRNA expression according to RT-qPCR analyses. F(8,18) = 6.09. D. ACE2 activity. F(8,18) = 26.34. E. TMPRSS2 mRNA expression according to RT-qPCR analyses. F(8,18) = 43.22. One-way ANOVA, followed by Tukey post-test. *p < 0.05. (n = 3/group).

Fig. 4

Evaluation of proteins related to autophagy flux, apoptosis and mitochondrial oxidative metabolism. Protein levels were analyzed by Western blot, and histograms are reported as the mean ± SD of protein levels after normalization with the average intensity of the bands from three independent experiments. A. LC3-II, F(8,15) = 4,2. B. Lamp-1, F(7,16) = 12.95. C. Total caspase-3 total, F(7,16) = 39.13. D. Cathepsin-L, F(8,10) = 9.1. E. ATP5H, F(8,17) = 12.73. F. Citrate synthase, F(8,16) = 2.26. G. Cytochrome c oxidase subunit 2 (COX-2), F(8,18) = 19.33. H. NADH dehydrogenase iron-sulfor protein 2 (NDUFS2), F(8,18) = 7.3. One-way ANOVA, followed by Tukey's post-test. *p < 0.05. (n = 3/group).

Fig. 5

PCA score plot was generated in Metaboanalyst v5.0 using A. ACE2, TMPRSS2, LC3II, LAMP1, Cathepsin-L, caspase-3, ATP synthase, citrate synthase, COX2, and NDUFS2, or B. ATP synthase, citrate synthase, COX2 and NDUFS2 (four proteins involved in energy metabolism). Green dots and clouds correspond to the cell lines with high replication capacity and 95 confidence intervals. Red dots and clouds indicate the cell lines with low replication capacity and 95 confidence intervals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Molecular mechanism related to SARS-CoV-2 replication. Created with Biorender.com

Fig. 7

ACE2 overexpression in pulmonary cells. A. Evaluation of ACE2 gene overexpression in BEAS-2B and A549 cells at 2, 30 and 45 days after nucleofection. F(2,4) = 6.8. B. ACE2 protein levels expression. F(3,8) = 64.94. C. ACE2 activity. F(3,14) = 229,3. Mixed effect model (REML), followed by Tukey's post-test. *p < 0.05.

Fig. 8

MTT Cell proliferation assay with ACE2 overexpressing cells. Comparative evaluation of proliferation between BEAS-2B and BEAS-2B-ACE2 strains after 1, 24, 48, 72 and 96 h of incubation. A. and C. Graphical data plot analysis of the proliferation assay. F(4,84) = 106.6, F(4,151) = 228.8. B. and D. Representative images of cells overexpressing ACE2 versus control cells (20× magnification). Two-way ANOVA, followed by Sidak's post-test. *p < 0.05.

Fig. 9

ACE2 overexpression alters the viral load in pulmonary cells. Viral load 2, 24, 48, 72 and 96 h after SARS-CoV-2 infection with MOI 0.2. SARS-CoV-2 extracellular. A. and intracellular. B. replication over time in BEAS-2B and A549 cell lines overexpressing ACE2 versus controls. Mixed effect model (REML), followed by the Bonferroni post-test. *p ≤ 0.05. F(4,5) = 1.1 (A), F(3,6) = 8.72, F(4,34) = 60.23 (C), F(3,16) = 5.9 (D).

Fig. 10

ACE2 overexpression increased pulmonary cell SARS-CoV-2 replication capacity. Created with Biorender.com