Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Abstract

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

Human Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry

“…Finally, we examined whether the SARS-CoV-2 inhibitory effect of defensins was cell-type-dependent. Intestinal epithelial cells (Caco-2) express moderate levels of endogenous hACE2, and lung epithelial cells (A549) have near undetectable levels of hACE2 but a high abundance of SARS-CoV-2 alternative receptors, CD147 and tyrosine-protein kinase receptor UFO (AXL) [ 28 ]. We found that HNP1, HD5 and RC101 exhibited anti-SARS-CoV-2 activity in both Caco-2 and A549 cells ( Figure 8 B), indicating that the inhibitory effect of defensins on viral infection was not receptor-specific or cell-type-dependent.…”

“…Since RC101 is known to inhibit Japanese encephalitis virus protease [ 51 ], investigations into the effects of defensins on serine protease are warranted. Additionally, alternative receptors including CD147 and AXL for SARS-CoV-2 viral entry have been reported, particularly in cells with a low abundance of hACE2 [ 28 , 52 , 53 ]. We have shown that pseudotyped viruses expressing SARS-CoV-2 spike proteins enter A549 cells through CD147 in a spike RBD-independent manner [ 28 ].…”

“…Additionally, alternative receptors including CD147 and AXL for SARS-CoV-2 viral entry have been reported, particularly in cells with a low abundance of hACE2 [ 28 , 52 , 53 ]. We have shown that pseudotyped viruses expressing SARS-CoV-2 spike proteins enter A549 cells through CD147 in a spike RBD-independent manner [ 28 ]. Similarly, AXL mediates SARS-CoV-2 infection of lung epithelial cells through interactions with the N-terminal domain of SARS-CoV-2 spike proteins [ 53 ].…”

“…Replication-defective HIV-1 luciferase-expressing reporter viruses pseudotyped with SARS-CoV-2 S proteins were produced by co-transfection of a plasmid encoding the envelope-deficient HIV-1 NL4-3 virus with the luciferase reporter gene (pNL4-3.Luc.R-E- or pNL4-3.Luc.R+E- from N. Landau, New York University) and a pcDNA3.1 plasmid expressing the SARS-CoV-2 glycoprotein into HEK 293T cells, which were seeded at 6–7 × 10 6 in a 10-cm dish and cultured overnight, using Lipofectamine 3000 (Thermo Fisher Scientific) as described previously [ 28 ]. The supernatant was collected 48 h after transfection and filtered.…”

CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 and its variants delta, alpha, beta, and gamma

“… 18 Other laboratory also reported the involvement of CD147 in SARS-CoV-2 viral entry. 19 However, whether CD147 can mediate cellular entry of SARS-CoV-2 variants remains unclear. In addition, whether CD147, as a signaling transducer, plays a role in the inflammation of COVID-19 diseases and contributes to cytokine storm in severe cases is unknown.…”