key: cord-0796655-fuxs1axy
authors: Davanzo, G. G.; Codo, A. C.; Brunetti, N. S.; Boldrini, V. O.; Knittel, T. L.; Monterio, L. B.; de Moraes, D.; Ferrari, A. J. R.; de Souza, G. F.; Muraro, S. P.; Profeta, G. . S.; Wassano, N. S.; Santos, L. N.; Carregari, V. . C.; Dias, A. . H. S.; Virgilio-da-Silva, J. V.; Castro, I.; Silva-Costa, L. . C.; Palma, A.; Mansour, E.; Ulaf, R. G.; Bernardes, A. F.; Nunes, T. A.; Ribeiro, L. C.; Agrela, M. V.; Moretti, M. L.; Buscaratti, L. I.; Crunfli, F.; Ludwig, R. . G.; Gerhardt, J. A.; Seste-Costa, R.; Forato, J.; Amorin, M. . R.; Texeira, D. A. T.; Parise, P. L.; Martini, M. C.; Bispo-dos-San,
title: SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes
date: 2020-09-28
journal: nan
DOI: 10.1101/2020.09.25.20200329
sha: 76a0749f2da61cfbecda1debfef50f3262d7e4b6
doc_id: 796655
cord_uid: fuxs1axy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects the lungs and may cause several immune-related complications such as lymphocytopenia and cytokine storm which are associated with the severity of the disease and predict mortality . The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is not fully understood. Here we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells in a mechanism that also requires ACE2 and TMPRSS2. Once inside T helper cells, SARS-CoV-2 assembles viral factories, impairs cell function and may cause cell death. SARS-CoV-2 infected T helper cells express higher amounts of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may explain the poor adaptive immune response of many COVID- 19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID- 19) .

SARS-CoV-2 infects the lungs and may cause several immune-related complications such as lymphocytopenia and cytokine storm which are associated with the severity of the disease and predict mortality 1, 2 . The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is not fully understood. Here we show that SARS-CoV-2 infects human CD4 + T helper cells, but not CD8 + T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells in a mechanism that also requires ACE2 and TMPRSS2. Once inside T helper cells, SARS-CoV-2 assembles viral factories, impairs cell function and may cause cell death. SARS-CoV-2 infected T helper cells express higher amounts of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may explain the poor adaptive immune response of many COVID-19 patients.

Coronavirus disease 2019 (COVID- 19) , which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerge in Wuhan in December of 2019. COVID-19 rapidly spread across the globe 3, 4 , being declared a pandemic by the WHO in March 11 th .

COVID-19 has caused nearly one million deaths around the world as of September 20 th , 2020.

Most of the deaths are associated with acute pneumonia, cardiovascular complications, and organ failure due to hypoxia, exacerbated inflammatory responses and widespread cell death 1, 5 . Individuals that progress to the severe stages of COVID-19 manifest marked alterations in the immune response characterized by reduced overall protein synthesis, cytokine storm, lymphocytopenia and T cell exhaustion [6] [7] [8] . In addition to these acute effects on the immune system, most convalescent individuals present low titres of neutralizing antibodies 9 . Moreover, the levels of antibodies against SARS-CoV-2 decay rapidly after recovery 10 , suggesting that SARS-CoV-2 infection may exert profound and long-lasting complications to adaptive immunity. In this context, one question that remains to be answered is how SARS-CoV-2 exert these effects on the immune system.

To infect cells, the spike glycoprotein of SARS-CoV-2 (sCoV-2) binds to the host angiotensinconverting enzyme 2 (ACE2), after which it is then cleaved by TMPRSS2 11 . While TMPRSS2 is ubiquitously expressed in human tissues ( fig. S1 ), ACE2 is mainly expressed in epithelial . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint and endothelial cells, as well as in the kidney, testis and small intestine ( fig. S1 ). Still, a wide variety of cell types are infected by SARS-CoV-2 [12] [13] [14] , even though some of these cells express very low levels of ACE2. We showed that this is the case for lymphocytes ( fig. S2 ). This finding suggests that SARS-CoV-2 has either an alternative mechanism to enter the cells or that auxiliary molecules at the plasma membrane may fix the virus until it interacts with an ACE2 molecule.

Since the structures of the spike of SARS-CoV-1 (sCoV-1) and the sCoV-2 proteins are similar 15, 16 , we used the P-HIPSTer algorithm to uncover human proteins that putatively interact with the viruses 17 . Seventy-one human proteins were predicted to interact with sCoV-1 ( fig. S3 ). We then cross-referenced the proteins with five databases of plasma membrane proteins to identify the ones located on the cell surface (see Methods for details). CD4 was the only protein predicted to interact with sCoV-1 that appeared in all five databases ( fig. S3 ).

CD4 is expressed mainly in T helper lymphocytes and has been shown to be the gateway for HIV 18 . Since CD4 + T lymphocytes orchestrate innate and adaptive immune responses 19, 20 , infection of CD4 + T cells by SARS-CoV-2 might explain lymphocytopenia and dysregulated inflammatory response in severe COVID-19 patients. Moreover, from an evolutionary perspective, the infection of CD4 + T cells represents an effective mechanism for viruses to escape the immune response 21 .

To test whether human primary T cells are infected by SARS-CoV-2, we purified CD3 + CD4 + and CD3 + CD8 + T cells from the peripheral blood of non-infected healthy controls/donors (HC), incubated these cells with SARS-CoV-2 for 1h, and then exhaustively washed them to remove any residual virus. The viral load was measured 24h post-infection. We were able to detect SARS-CoV-2 RNA in primary CD4 + T cells but not CD8 + T cells ( fig. 1A) . To confirm the presence of SARS-CoV-2 infection, we performed in situ hybridization using probes against the viral RNA-dependent RNA polymerase (RdRp) gene, immunofluorescence for sCoV-2 and transmission electron microscopy. All three approaches confirmed that SARS-CoV-2 infects CD4 + T cells ( fig. 1B, 1C and fig. S4 ). Moreover, we detected different SARS-CoV-2 RNAs in infected CD4 + T cells ( fig. S5A) . Notably, the viral RNA level increases with time ( fig. 1D) and we identified the presence of the negative strand (antisense) of SARS-CoV-2 in the infected cells ( fig. 1E) , demonstrating that the virus is able to assemble viral factories and replicate in T helper cells. Plaque assay also revealed that SARS-CoV-2-infected CD4 + T cells produce infectious viral particles ( fig. 1F and S5B-C) .

To confirm that SARS-CoV-2 infects CD4 + T cells in vivo, we purified CD4 + and CD8 + T cells from peripheral blood cells of COVID-19 patients (table S1). Similar to our ex vivo findings, SARS-CoV-2 RNA was detected in CD4 + T cells, but not in CD8 + T cells from COVID-19 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . patients ( fig. 1G ). Yet, the viral load was markedly higher in CD4 + T cells from severe COVID-19 patients in comparison to patients with the moderate form of the disease ( fig. 1G ). Using publicly available single-cell sequencing data 22 , we were also able to detect the presence of SARS-CoV-2 RNA in 2.1% of CD4 + T cells of the bronchoalveolar lavage (BAL) of patients with the severe but not the moderate form of COVID-19 ( fig. 1H) . Thus, our data demonstrate that SARS-CoV-2 infects CD4 + T cells and the infection associates with the severity of COVID-

We sought to explore the role of the CD4 molecule in SARS-CoV-2 infection. Based on the putative interaction found using P-HIPSTer, we performed molecular docking analyses and predicted that sCoV-2 receptor binding domain (RBD) directly interacts with the N-terminal domain (NTD) of CD4 Ig-like V type ( fig. 2A and S6 ). Molecular dynamics simulations with stepwise temperature increase were applied to challenge the kinetic stability of the docking model representatives ( fig. 2B ). Two models remained stable after the third step of simulation at 353 Kelvin and represent likely candidates for the interaction between the CD4 NTD and sCoV-2 RBD ( fig. 2B) . Additionally, convergence towards the two surviving models was tested for closely related binding mode models present among the remaining cluster candidates and was verified in one case, which indicates plausible and rather stable interaction between CD4 NTD and sCoV-2 RBD ( fig. S6) . The interaction region of RBD is predicted to overlap with that of human ACE2 ( fig. 2C and 2D) . The interaction region of RBD is predicted to overlap with that of human ACE2 ( fig. 2C and 2D ). The interaction between CD4 and sCoV-2 was confirmed by co-immunoprecipitation of sCoV-2 and full length recombinant CD4 ( fig. 3A ).

Consistent with a mechanism where CD4-sCoV-2 interaction is required for infection, we To gain further insights into the importance of CD4-sCoV-2 binding to SARS-CoV-2 infection, we purified CD4 + T cells and pre-treated them with anti-CD4 polyclonal antibody. We observed a dose-dependent reduction in viral load in CD4 + T cells pre-treated with anti-CD4 antibody, showing that CD4 is necessary for SARS-CoV-2 infection ( fig. 3C) . Remarkably, the same monoclonal antibody that has been used to block HIV entry in CD4 + T cells 18,23 also blocked SARS-CoV-2 entry in a dose dependent manner ( fig. 3D ). This observation is consistent with our in silico model ( fig. 2 and S6E ) that predicts that sCoV-2 binds to a region of CD4 which is neighbor to where envelope-displayed glycoprotein spike complex (Env) is shown to bind 24 . These data demonstrate that the CD4 molecule is necessary for infection of CD4 + T cells by SARS-CoV-2 and suggest that SARS-CoV-2 may use a mechanism that somehow resembles HIV infection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09. 25.20200329 doi: medRxiv preprint In HIV infection, CD4 alone is not sufficient to allow the virus to enter CD4 + T cells 25 . Instead, the Env must also interact with co-receptors (CCR5 or CXCR4) 25, 26 . In this context, we tested whether CD4 alone was sufficient to allow SARS-CoV-2 entry. Inhibition of ACE2 using polyclonal antibody abrogated SARS-CoV-2 entry in CD4 + T cells ( fig. 3E) , suggesting that the canonical entry mechanism involving ACE2 and TMPRSS2 11 is also required. To exclude the possibility that the polyclonal anti-ACE2 antibody cross-reacts with CD4, we designed a peptide to specifically block ACE2-sCoV-2 interaction (fig. S7) . The peptide recapitulated the effect of anti-ACE2 antibody and also reduced the viral load in a dose dependent manner ( fig.   3F ). Similarly, inhibition of TMPRSS2 with camostat mesylate also reduced SARS-CoV-2 infection ( fig. 3G) . Hence, ACE2, TMPRSS2 and CD4 act in concert to allow the infection of CD4 + T cells by SARS-CoV-2.

To assess the consequences of SARS-CoV-2 infecting CD4 + T cells, we performed mass spectrometry-based shotgun proteomics in ex vivo infected CD4 + T cells. We found that SARS-CoV-2 infection affects multiple housekeeping pathways associated with the immune system, infectious diseases, cell cycle and cellular metabolism ( fig. 4A, 4B The infection of CD4 + T cells by HIV also causes an increase in IL-10 production 28, 29 . The expression and release of IL-10 has been widely associated with chronic viral infections and determines viral persistence 30 . Noteworthy, increased serum levels of IL-10 are associated with COVID-19 severity 31, 32 . We found that IL10 expression by CD4 + T cells was higher in BAL is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint Upregulation of IL10 in HIV-infected cells requires STAT3-independent activation of the transcription factor CREB-1 via Ser 133 phosphorylation 33 . Consistent with the similarities between SARS-CoV-2 and HIV infections, CREB-1 phosphorylation at Ser 133 was increased in SARS-CoV-2-infected CD4 + T cells ( fig. 4D and S10E) . Thus, SARS-CoV-2 infection appears to directly trigger a signaling cascade that culminates in upregulation of IL10 in CD4 + T cells. Indeed, expression of IL10 is positively correlated with viral load in circulating CD4 + T cells from COVID-19 patients (fig. 4E) . Altogether, our data demonstrate that SARS-CoV-2 infects CD4 + T cells, impairs cell function, leads to increased IL10 expression and compromises cell viability, which in turn dampens immunity against the virus and contributes to disease severity. IL-10 is a powerful anti-inflammatory cytokine and has been previously associated with viral persistence 30 . Serum levels of IL-10 increase during the early stages of the disease -when viral load reaches its peak -and may predict COVID-19 outcome 31, 32 . This increase occurs only in patients with the severe form of COVID-19 32 . Consistent with these findings, we found that expression of IL10 positively correlates with viral load in CD4 + T cells. This is an unique feature of patients with the severe form of COVID-19, since we could not detect the virus in CD4 + T cells from patients with the moderate form of the disease and IL10 expression in CD4 + T cells is much lower in these patients. In contrast, we found IFNG and IL17A to be upregulated in CD4 + T cells of patients with the moderate illness, indicating a protective role for these cytokines. However, in patients with the severe symptoms, the expression of IFNG and IL17A in CD4 + T cells is dampened. IL-10 is a known suppressor of Th1 and Th17 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint responses 36 and it is likely to contribute to the changes in IFNG and IL17A. These features will ultimately reflect in the quality of the immune response, which in combination with T cell death and consequent lymphopenia, may result in transient/acute immunodeficiency and impair adaptive immunity in severe COVID-19 patients [6] [7] [8] .

How long these alterations in T cell function persist in vivo and whether they have long-lasting impacts on adaptive immunity remains to be determined. Hence, avoiding T cell infection by blocking sCoV-2-CD4 interaction and boosting T cell resistance against SARS-CoV-2 might represent complementary therapeutic approaches to preserve immune response integrity and prevent patients from progressing to the severe stages of COVID-19. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint 2 and CD4 NTD interaction obtained by alignment of sCoV-2 RBD from model 148 to the sCoV-2 RBD opened state from PDB 6vyb EM structure. ***p < 0.001, **p < 0.00, *p < 0.05 compared to all. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 28, 2020. . https://doi.org/10.1101/2020.09.25.20200329 doi: medRxiv preprint

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