SARS-CoV-2 infects human CD4+T helper cells by binding to the CD4 molecule

The authors from Brazil investigated the replication capacity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the effects of replication on various immune cells, especially those involved in the formation of immunological memory and an effective adaptive response, such as CD4+T lymphocytes. The results showed that SARS-CoV-2 infects CD4+T helper cells, but not CD8+T cells, and that SARS-CoV-2 spike glycoprotein (S) binds to the CD4 molecule directly, which in turn mediates the entry of virus in T helper cells. According to researchers, CD4 stabilizes SARS-CoV-2 on the cell membrane until the virus encounters the host angiotensin-converting enzyme 2 (ACE2) to enter the cell.

It has been suggested that SARS-CoV-2 infection is caused by the S glycoprotein binding to ACE2, after which it is cleaved by TMPRSS2. ACE2 is mainly expressed in epithelial and endothelial cells, as well as in the kidney, testis, and small intestine. Since a wide variety of cell types that express very low levels of ACE2 are also infected by SARS-CoV-2, it shows that binding of S glycoprotein to other cell surface proteins facilitates viral entry.

About the study

In order to determine whether human primary T cells are infected with SARS-CoV-2, the authors purified CD3+CD4+ and CD3+CD8+T cells from the peripheral blood of uninfected healthy donors, and incubated these cells with SARS-CoV-2 for 1 hour. 24 hours post infection, the viral load was measured. SARS-CoV-2 RNA was detected in CD4+T cells, but not CD8+T cells.

To confirm the presence of SARS-CoV-2 in T cells, researchers performed in situ hybridization using probes against the viral RNA-dependent RNA polymerase gene, immunofluorescence for SARS-CoV-2 S protein using antibodies against the S protein, and transmission electron microscopy. Additionally, primary CD4+T cells were infected with the SARS-CoV-2 pseudotype virus. All approaches confirmed that SARS-CoV-2 infected CD4+T cells.

The identification of the negative strand (antisense) of SARS-CoV-2 in infected cells demonstrated that the virus replicated in T helper cells. A plaque assay analysis demonstrated that CD4+T cells infected with SARS-CoV-2 released infectious viral particles, but much less efficiently than Vero cells (positive control).

The scientists then performed molecular docking analyzes to investigate the role of the CD4 molecule in SARS-CoV-2 infection. They predicted that the receptor binding domain (RBD) of the S protein directly interacts with the N-terminal domain (NTD) of the CD4. The findings revealed a stable and plausible interaction between the CD4 NTD and the SARS-CoV-2 RBD.

Co-immunoprecipitation confirmed the interaction between the CD4 and the S protein. A fluorescence anisotropy assay confirmed the high-affinity interaction between the RBD of the S protein and the CD4 molecule, and between the full-length S protein and the CD4 molecule. These results suggest that the interaction interface between the SARS-CoV-2 S protein and the CD4 molecule occurs at the RBD. 

The researchers then assessed the importance of interaction between the S protein and CD4 for the  SARS-CoV-2 infection. They purified human CD4+T cells and incubated them with a CD4 monoclonal antibody. The inhibition of CD4 led to a reduction in the SARS-CoV-2 load. In human T-cell lines expressing CD4, the presence of CD4 was sufficient to increase viral load compared with T cell lines not expressing CD4. The SARS-CoV-2 pseudotype model confirmed these results.

Further, the research team examined whether CD4 alone would be sufficient to allow SARS-CoV-2 entry, given a very low expression of ACE2 in CD4+T cells. The inhibition of ACE2 by a polyclonal antibody diminished entry of SARS-CoV-2 in CD4+T cells. Also, the inhibition of TMPRSS2 by camostat mesylate resulted in reduction of the SARS-CoV-2 load. These results indicate that ACE2, TMPRSS2, and CD4 are all required for the infection of CD4+T cells with SARS-CoV-2.

To evaluate the consequences of T cell infection with SARS-CoV-2, mass spectrometry-based shotgun proteomics was performed in CD4+T cells exposed to SARS-CoV-2 ex vivo. The results revealed alterations in multiple pathways associated with stress response, cellular metabolism, apoptosis, and cell cycle regulation. In addition, the ex vivo exposure of CD4+T cells to SARS-CoV-2 decreased cell viability by 10% 24 hours after the infection.

The authors also assessed the expression of key pro- and anti-inflammatory cytokines involved in the immune response elicited by CD4+T cells. SARS-CoV-2 increased interleukin (IL)-10 expression in CD4+T cells. Interestingly, the expression of interferon (IFN)-γ and IL-17A decreased in CD4+T cells from patients with severe disease, and increased in CD4+T cells from patients with moderate disease. 

In conclusion, this study showed that SARS-CoV-2 infects CD4+T helper cells, and that S protein binds directly to the CD4 molecule. The mechanism of infection involves the interaction between the RBD domain of the S protein and the NTD of the CD4 molecule, and entry is mediated by the canonical ACE2/TMPRSS2 pathway.

The findings further demonstrated that SARS-CoV-2 impaired T cell function, led to increased IL-10 expression, and compromised cell viability. Thus, immune response dysregulation associated with severe COVID-19 is triggered, at least in part, by infection of CD4+T helper cells with SARS-CoV-2. The authors also argue that, from an evolutionary perspective, infection of CD4+T helper cells by SARS-CoV-2 is an effective mechanism for the virus to evade the immune response. It remains to be elucidated for how long these changes in T cell function persist in vivo and whether they have long-lasting impacts on adaptive immunity.

This article was published in eLife.

Journal Reference

Brunetti NS, Davanzo GG, de Moraes D, Ferrari AJR, Souza GF, Muraro SP, Knittel TL, Boldrini VO, Monteiro LB, Virgílio-da-Silva JV et al. SARS-CoV-2 uses CD4 to infect T helper lymphocytes. eLife 2023; 12:e84790.