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Tag: ACE2

  • Study reveals how SARS-CoV-2 hijacks lung cells to drive COVID-19 severity

    Study reveals how SARS-CoV-2 hijacks lung cells to drive COVID-19 severity

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    In a recent study published in the Journal of Experimental Medicine, researchers identified the cellular tropism and transcriptome consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by infecting human lung tissue and using single-cell ribonucleic acid sequencing (scRNA-seq) to rebuild the transcriptional program in “infection pseudotime” for distinct lung cell types.

    Lower respiratory infections, such as coronavirus disease 2019 (COVID-19), are a leading cause of death worldwide, producing pneumonia and acute respiratory distress syndrome. Understanding their early phases is difficult. Researchers used classical histopathological approaches and single-cell multi-omic profiling to infer early phases in human pathogenesis from lung lavage, biopsy, or autopsy materials. These approaches reveal a thorough picture of COVID-19 pneumonia at unparalleled cellular and molecular resolution, implying infection models including alveolar epithelium, capillaries, macrophages, and myeloid cells.

    Study: Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung. Image Credit: Dotted Yeti / ShutterstockStudy: Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung. Image Credit: Dotted Yeti / Shutterstock

    About the study

    In the present study, researchers developed an experimental COVID-19 model to investigate early molecular processes and pathogenic mechanisms of SARS-CoV-2 infection at the cellular level in native tissues of the human lung.

    The researchers established SARS-CoV-2’s cellular tropism and its unique and dynamic impacts on host cellular gene expression in specific types of lung cells. Prominent targets were lung-resident macrophages, of which one SARS-CoV-2 takes over transcriptomes, inducing a targeted host interferon (IFN) antiviral program, and several chemokines and pro-fibrotic and pro-inflammatory and cytokines signaling to various structural and immunological cells of the lung.

    To determine the early stages of COVID-19 in human lungs, the researchers sliced lung tissue obtained from surgical specimens or organ donor individuals into thick sections and used them for tissue culture analysis. Subsequently, they exposed the tissues to the SARS-CoV-2 USA-WA1 2020 strain at 1.0 multiplicity of infection (MOI) for two hours before allowing the SARS-CoV-2 infection to continue for two to three days. They performed a plaque test on culture supernatants.

    The researchers separated the slices and examined them by scRNA-seq to evaluate host and viral genetic expression during the SARS-CoV-2 infection. They also examined the viral RNA molecules’ junctional structure and processing by analyzing the scRNA-seq dataset with the SICILIAN framework. They used molecular atlas markers to distinguish lung cell types in healthy lung slices and measure viral RNA levels in infected cells.

    The team performed multiplexed single-molecule fluorescence in situ hybridization (smFISH) to confirm lung cell tropism findings and show infected cells. They used single-cell gene expression patterns to identify cellular targets for inflammatory and pro-fibrotic signals elicited by the SARS-CoV-2 infection of a-IMs. They devised a technique for purifying macrophage populations from human lungs with a SARS-CoV-2 spike (S) protein-pseudotyped lentivirus (lenti-S-NLuc-tdT) to investigate lung macrophage entrance routes.

    The researchers productively infected human lung slices cultivated ex vivo with SARS-CoV-2, with production rising between 24 and 72 hours of culture. They heat-inactivated, ultraviolet (UV)-treated, or administered 10.0 µM remdesivir, an RNA-dependent RNA polymerase inhibitor used as a COVID-19 therapeutic, to prevent viral stock infection.

    Results

    The analysis showed that SARS-CoV-2 preferentially infects active interstitial macrophages (IMs), which can amass hundreds of SARS-CoV-2 RNA molecules, comprising >60% of the cell transcriptome and producing dense viral RNA bodies. Infected alveolar macrophages (AMs) exhibit no severe reactions, with spike (S) protein-dependent viral entrance into AMs utilizing angiotensin-converting enzyme 2 (ACE2) and the cluster of differentiation 169 (CD169) and IM entry via CD209.

    They found canonical sub-genomic junctions between the unusual sequence reads beyond their 39 terminal regions, indicating canonical-type SARS-CoV-2 messenger RNA (mRNA) production in the pulmonary cultures. They also found hundreds of new subgenomic junctions, showing a wide range of non-canonical and canonical sub-genomic SARS-CoV-2 RNAs produced during pulmonary infection.

    Model of initiation, transition, and pathogenesis of COVID-19 and the viral lifecycle in AMs and IMs. (a–d) Model of COVID-19 initiation in the human lung and transition from viral pneumonia to lethal COVID-19 ARDS. (a) SARS-CoV-2 virion dissemination and arrival in the alveoli. Luminal AM encounter virions shed from the upper respiratory tract that enter the lung. AMs can express low to moderate numbers of viral RNA molecules and can propagate the infection but “contain” the viral RNA from taking over the total transcriptome and show only a very limited host cell inflammatory response to viral infection. (b) Replication and epithelial injury. SARS-CoV-2 virions enter AT2 cells through ACE2, its canonical receptor, and “replicate” to high viral RNA levels, producing infectious virions and initiating viral pneumonia. (c) a-IM takeover and inflammation signaling. SARS-CoV-2 virions spread to the interstitial space through either transepithelial release of virions by AT2 cells or injury of the epithelial barrier, and enter a-IMs. Infected a-IMs can express very high levels of viral RNA that dominate (“take over”) the host transcriptome and can propagate the infection. Viral takeover triggers induction of the chemokines and cytokines shown, forming a focus of inflammatory and fibrotic signaling. (d) Endothelial breach and immune infiltration. The a-IM inflammatory cytokine IL6 targets structural cells of the alveolus causing epithelial and endothelial breakdown, and the inflammatory cytokines recruit the indicated immune cells from the interstitium or bloodstream, which flood and infiltrate the alveolus causing COVID-19 ARDS. Local inflammatory molecules are amplified by circulating immune cells, and reciprocally can spread through the bloodstream to cause systemic symptoms of cytokine storm. (e) Comparison of the SARS-CoV-2 viral lifecycle in AMs and IMs. Although both can produce infectious virions, note differences in viral entry receptors (AMs can use ACE2 and CD169/SIGLEC1, whereas IMs use CD209); viral RNA transcription of dsRNA intermediates (greater in AMs); replication of full-length genomic RNA (greater in IMs); viral takeover, formation of RNA bodies, and induction of a robust host cell inflammatory response (only in IMs), and cell destruction/death (only in IMs).Model of initiation, transition, and pathogenesis of COVID-19 and the viral lifecycle in AMs and IMs. (a–d) Model of COVID-19 initiation in the human lung and transition from viral pneumonia to lethal COVID-19 ARDS. (a) SARS-CoV-2 virion dissemination and arrival in the alveoli. Luminal AM encounter virions shed from the upper respiratory tract that enter the lung. AMs can express low to moderate numbers of viral RNA molecules and can propagate the infection but “contain” the viral RNA from taking over the total transcriptome and show only a very limited host cell inflammatory response to viral infection. (b) Replication and epithelial injury. SARS-CoV-2 virions enter AT2 cells through ACE2, its canonical receptor, and “replicate” to high viral RNA levels, producing infectious virions and initiating viral pneumonia. (c) a-IM takeover and inflammation signaling. SARS-CoV-2 virions spread to the interstitial space through either transepithelial release of virions by AT2 cells or injury of the epithelial barrier, and enter a-IMs. Infected a-IMs can express very high levels of viral RNA that dominate (“take over”) the host transcriptome and can propagate the infection. Viral takeover triggers induction of the chemokines and cytokines shown, forming a focus of inflammatory and fibrotic signaling. (d) Endothelial breach and immune infiltration. The a-IM inflammatory cytokine IL6 targets structural cells of the alveolus causing epithelial and endothelial breakdown, and the inflammatory cytokines recruit the indicated immune cells from the interstitium or bloodstream, which flood and infiltrate the alveolus causing COVID-19 ARDS. Local inflammatory molecules are amplified by circulating immune cells, and reciprocally can spread through the bloodstream to cause systemic symptoms of cytokine storm. (e) Comparison of the SARS-CoV-2 viral lifecycle in AMs and IMs. Although both can produce infectious virions, note differences in viral entry receptors (AMs can use ACE2 and CD169/SIGLEC1, whereas IMs use CD209); viral RNA transcription of dsRNA intermediates (greater in AMs); replication of full-length genomic RNA (greater in IMs); viral takeover, formation of RNA bodies, and induction of a robust host cell inflammatory response (only in IMs), and cell destruction/death (only in IMs).

    Heat, UV-C inactivation, or remdesivir therapy prevented the development of canonical and non-canonical connections. The team observed SARS-CoV-2 takeover of an activated IM subtype in 176,382 cells with high-quality transcriptomes obtained from infected lung slices of four donor lungs and in 112,359 cells from mock-infected slices (cultured without viral addition) and 95,389 uncultured control cells (directly from freshly cut lung slices). A differential gene expression study of a-IMs over infection pseudotime revealed host gene expression alterations corresponding to SARS-CoV-2 RNA levels.

    The study found that COVID-19 pneumonia infection and takeover cause an early antiviral cell response specific to activated interstitial macrophages, resulting in a powerful immunological and fibrotic signaling center. Inflammasome activation is uncommon and only detectable late in a-IM infection. Blocking antibodies against CD169 and CD209 prevented entrance into IMs and AMs. The study also highlighted IMs as the most vulnerable lung target, with initial emphasis on inflammation and fibrosis. Two unique molecular lineages of macrophage targets react differently to SARS-CoV-2, influencing etiology and treatments.

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  • Severity of current SARS-CoV-2 variants is not linked to the number of mutations

    Severity of current SARS-CoV-2 variants is not linked to the number of mutations

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    New research from UNC Charlotte’s Center for Computational Intelligence to Predict Health and Environmental Risks (CIPHER) has found that the two most prevalent strains of the virus that cause COVID-19, SARS-CoV-2 variants BA.2.86 and JN.1, are not significantly better than their predecessor Omicron at evading immune responses and causing infections despite having a high number of mutations compared to previous variants.

    When first identified, Omicron offshoots BA.2.86 and its close relative JN.1 raised significant public health concerns. These concerns were tied to the fact that the original Omicron variant was highly mutated, resulting in both immune evasion and breakthrough infection, as well as more infectious and highly-mutated compared to earlier variants.

    There was some speculation that large numbers of new mutations in BA.2.86 and JN.1 conferred a greater ability of these variants to evade the human immune system and be more transmissible. Extensive computational analyses conducted by a team of UNC Charlotte scholars and students determined that these variants only had small, statistically insignificant changes in immune evasion and transmissibility infection capacity compared to earlier variants, including Omicron.

    These results really surprised me. The fact that Omicron, with its large set of mutations, led to greater immune evasion and a surge in cases and hospitalizations was predictable. However, BA.2.86 and JN.1 have yet another large set of mutations, and while we have seen some signals of increased prevalence of these two variants in wastewater and genomic surveillance, there has not been an accompanying large surge in cases or hospital burden.”


    Daniel Janies, Co-Director of CIPHER and the Carol Grotnes Belk Distinguished Professor of Bioinformatics and Genomics in the College of Computing and Informatics

    To assess the immune evasion of BA.2.86 and JN.1, the UNC Charlotte research team performed an extensive in silico analysis on the Receptor Binding Domain (RBD; the region of the viral genome against which vaccines are designed) of SARS-CoV-2, comparing the two newer variants to previous variants to calculate the relative binding affinity of neutralizing antibodies to the RBD from vaccinated patients, infected patients and therapeutic sources. In addition to antibody analysis, researchers calculated the relative binding affinity of BA.2.86 and JN.1 to Angiotensin Converting Enzyme-2 (ACE2) in comparison to previous variants.

    The team found minor changes in binding affinity for neutralizing antibodies and ACE2 for BA.2.86 and JN.1 in comparison to previous SARS-CoV-2 variants. However, those changes were not statistically significant. Therefore, they concluded that BA.2.86 and JN.1 have no significant increase in immune evasion or infection capacity to previous variants. In explaining their results, the researchers caution that genomic surveillance, which counts mutations or relative prevalence of a variant, does not necessarily reveal the functional and health impacts of the variant.

    In a study awaiting publication outlining their research, the team discusses the benefits of their approach to understand the function of variants and the need for future studies to assess variation outside of the RBD for future analysis. Future studies in this area will benefit from an increased focus on antibodies derived from memory B-cells that produce antibodies in response to SARS-CoV-2.

    “In patients whose immune systems have been exposed to a previous Omicron variant, memory B-cells may provide significant protection for the newer Omicron variants BA.2.86 and JN.1,” said Shirish Yasa, a current Charlotte bioinformatics and computer science senior who helped conduct this research. “This protection conferred by memory B-cell-derived antibodies is a process not yet well studied. An increase in Omicron targeting memory B-cells via vaccination and prior infection could be a significant factor in the overall reductions we have seen in hospitalizations and deaths for patients exposed to the descendents of the original Omicron variant.”

    This research contributes to the functional understanding of SARS-CoV-2 variants BA.2.86 and JN.1, building on the studies of genomic surveillance. Moreover, this UNC Charlotte effort has introduced new methodologies for functional computational immunology, which will help in the ongoing efforts to mitigate the consequences of the COVID-19 pandemic.

    Source:

     University of North Carolina at Charlotte

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  • Scientists uncover a new doorway for SARS-CoV-2 into human cells

    Scientists uncover a new doorway for SARS-CoV-2 into human cells

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    In a recent study published in the journal Proceedings of the National Academy of Sciences, researchers demonstrated that human transferrin receptor (TfR) mediates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

    Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, presents influenza-like manifestations, including mild-to-severe pneumonia, acute respiratory distress syndrome, multiorgan failure, and fatal lung injury. Further, the etiology and pathogenesis of COVID-19 are not entirely understood and targeted therapies remain inadequate.

    The viral spike protein binds to the host receptor, angiotensin-converting enzyme 2 (ACE2), for cellular entry. Although SARS-CoV-2 preferentially infects cells in the respiratory tract, the virus has been detected in virtually all organs. Studies have revealed the presence of SARS-CoV-2 RNA in diverse cells lacking ACE2, suggesting that other receptors or co-receptors may mediate viral entry.

    Study: Human transferrin receptor can mediate SARS-CoV-2 infection. Image Credit: Kateryna Kon / ShutterstockStudy: Human transferrin receptor can mediate SARS-CoV-2 infection. Image Credit: Kateryna Kon / Shutterstock

    The study and findings

    In the present study, researchers identified TfR as an alternative receptor mediating the cellular entry of SARS-CoV-2. First, they used co-immunoprecipitation (Co-IP) to identify host proteins interacting with the viral spike in Calu-3 cells. This revealed 293 proteins, including 42 transmembrane proteins; two proteins were associated with entry (ACE2 and TfR). Next, the team evaluated TfR expression in the respiratory tract and liver in mice.

    TfR expression, both transcript and protein levels, was substantially higher in the lungs and trachea than in other tissues. Using immunohistochemical analysis, the researchers investigated the effects of SARS-CoV-2 on TfR expression in the lungs of humanized ACE2 (hACE2) mice and monkeys. This revealed a 1.5- and 1.8-fold increase in TfR expression in mice and monkeys, respectively.

    In addition, surface plasmon resonance revealed direct interactions between the viral spike and human TfR. Notably, the spike protein lacked interactions with Syrian hamster or mouse TfR. Docking analysis predicted two peptide sequences (QK8: QDSNWASK and SL8 SKVEKLTL) in TfR to be involved at the interface of TfR-spike interactions.

    Mutagenesis and Co-IP revealed that the A529 residue in TfR was essential for interactions with the spike. Further analysis indicated that physiological interactions between spike and TfR occurred at the cellular surface and during endocytosis. This was confirmed by electron microscopy using SARS-CoV-2 pseudoviral spike and HEK293/hACE2 and BHK-21/TfR cells.

    Next, the team evaluated the effects of soluble TfR, anti-TfR antibody, and SL8 and QK8 peptides on SARS-CoV-2 infection using reverse-transcription polymerase chain reaction (RT-PCR) and plaque assays. Results showed their inhibitory effects on SARS-CoV-2 in Vero E6 and Calu-3 cells. Cytotoxicity was not observed even at 1,000 nM.

    Confocal microscopy revealed that TfR was widespread on the surface of Calu-3 and Vero E6 cells, with the colocalization of TfR and SARS-CoV-2 at the surface and during endocytosis. Notably, treatment with the anti-TfR antibody inhibited the colocalization. Further, electron microscopy showed that viral particles were present in the cytosol and clathrin-coated pits in Vero E6 cells; likewise, treatment with anti-TfR antibody inhibited viral internalization.

    Next, ACE2 was knocked out (KO) from Calu-3 and Vero E6 cells and the cells were infected with SARS-CoV-2. This inhibited infection by 40% to 50%, suggesting that ACE2 might not be the only receptor mediating infection. In addition, TfR knockdown (KD) inhibited infection by 30%, whereas its overexpression (OE) promoted infection. TfR KO was not performed as it is lethal. TfR OE or KD did not impact ACE2 expression.

    Further, the team transfected C57 mice with adenovirus vector (Ad5) expressing hACE2 or humanized TfR (hTfR) and infected them with SARS-CoV-2. Viral load in the lungs in Ad5-hTfR and Ad5-hACE2 mice was significantly higher than in Ad5-empty mice. Finally, the researchers evaluated the effects of the anti-TfR antibody on infection in rhesus macaques. Anti-TfR antibody inhibited viral replication and reduced pneumonia.

    Viral load in the respiratory epithelium was also significantly lower between 3- and 7 days post-infection (dpi) compared to controls. Radiographs taken at 0 and 5 dpi revealed significantly less severe pulmonary infiltration in antibody-treated macaques relative to controls. Antibody-treated animals had no significant pulmonary lesions, while controls showed lung lesions of varying degrees.

    Conclusions

    Taken together, the study described the human TfR as a receptor for SARS-CoV-2. TfR can directly bind to the viral spike at an affinity comparable to that of ACE2. Notably, mouse TfR and the viral spike lacked interactions. Soluble TfR, SL8, and QK8 peptides and anti-TfR antibodies can inhibit infection. The team also illustrated the antiviral effects of the anti-TfR antibody in rhesus macaques. Overall, TfR could serve as an alternative infection pathway, facilitating viral entry through endocytosis.

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