SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 7

Figure 7. SARS-CoV-2 induces a positive feed-back loop of infection in mice.

A Schematic diagram of the human ACE2 knockin (hACE2 KI) mutant allele. The knockin with hACE2 cDNA plus SV40 late poly A signal results in blocking the transcription of mouse ACE2 (mACE2). The box with numbers, the exons of mACE2; arrowheads, the primers for genotyping PCR.

B-F Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Infection eﬃciencies of SARS-CoV-2 pseudovirus into mouse tissues were measured by IVIS and presented as luminescence counts at 4 days post infection (B). Immunoblotting of ACE2, GLK, and tubulin proteins in the lung tissues of infected or non-infected mice (C). Representative immunohistochemistry of the SARS-CoV-2 spike protein (in red) and GLK protein (in green) in the lung tissues of the infected hACE2 KI and wild-type mice (D). Scale bars, 20 μm (D). Immunoblotting of CD9 and mouse/human ACE2 in the exosomes isolated from the BALFs (E) of the infected mice. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. The hACE2-containing exosomes in the lung tissues of wild-type and hACE2 KI mice were determined by in situ proximity ligation assays (PLA) of close proximity (<40 nm) between hACE2 and the exosome marker CD9 using anti-human ACE2 antibody plus anti-CD9 antibody (F). Scale bars, 20 μm (F).

G Schematic depiction of the construction of the EF-1α promoter-driven, murine codon-optimized human ACE2 (hACE2) cDNA with bovine growth hormone (bGH) poly A signal.

H-J Human ACE2 transgenic (EF1α-hACE2 Tg) mice were intranasally infected with 2x105 pfu of live SARS-CoV-2. The survival rates of in EF1α-hACE2 Tg mice challenged with/without SARS-CoV-2 were monitored (H). The lung tissues of infected mice at day 3 days post infection were collected and then analyzed by immunohistochemistry (IHC) and PLA assays. Representative IHC data of hACE2 (in red) and GLK (in green) in the lung tissues of the infected wild-type and EF1α-hACE2 Tg mice were shown (I). The hACE2-containing exosomes in the lung tissues of infected wild-type and EF1α-hACE2 Tg mice were determined by PLA (J). Cell nuclei were stained with DAPI. Original magnification, ×630. Scale bars, 10 μm.

K, L Adoptive transfer of ACE2-containing exosomes facilitates SARS-CoV-2 pseudovirus infection. Serum exosomes (exo) isolated from wild-type (WT) mice and hACE2 KI/activated GLK transgenic (PolII-GLK E351K Tg) mice were adoptively transferred into wild-type recipient mice every 3 days for 12 days, followed by SARS-CoV-2 pseudovirus infection. Infection eﬃciencies into mouse tissues were measured by IVIS and presented as luminescence counts at 3 days post infection (K). hACE2-containing exosomes in the lung tissues of exosome-recipient wild-type mice were determined by PLA using anti-human ACE2 antibody plus anti-CD9 antibody (L). Scale bars, 10 μm (L).

Data information: Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. WT, wild-type mice; KI, hACE2 knockin mice. Tg, transgenic mice.

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