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SARS Coronavirus Spike Protein Expression in HL-CZ Human Promonocytic Cells: Monoclonal Antibody and Cellular Transcriptomic Analyses

ABSTRACT: The SARS coronavirus (CoV) spike protein is a target of intensive research, as it is a major virulence factor. Transfection of SARS-CoV spike into Vero E6, HEK293T and HL-CZ cells leads to strong expression of the glycosylated spike protein, as shown by Western blot analyses and immunofluorescent imaging using spike-specific human monoclonal antibodies, indicating the potential utility of these antigens and antibodies as diagnostic reagents. Furthermore, we employed cDNA microarray analysis to probe the changes in host gene transcription attributed to transfection of a codon-optimized spike construct into the HL-CZ cell line of monocyte lineage that is linked to immunological responses. A diverse representation of 100 genes displayed altered transcriptional patterns in response to SARS-CoV spike expression, with 61 upregulated and 39 downregulated genes. Genes involved in intracellular trafficking, signaling, modulation or transcription were generally upregulated. In contrast, genes involved in cell metabolism and cytoskeleton formation were notably downregulated. The transcripts of other functional categories exhibited varied responses to SARS-CoV spike transfection. Collectively, our analyses elucidate numerous and complex transcriptomic events that occur in response to spike protein expression and that contribute towards SARS-CoV pathogenesis.

SUBMITTER: Lal S

PROVIDER: S-EPMC7176169 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Project description:The ongoing COVID-19 pandemic is a clear and present threat to global public health. Research into how the causative SARS-CoV-2 virus together with its individual constituent genes and proteins interact with target host cells can facilitate the development of improved strategies to manage the acute and long-term complications of COVID-19. In this study, to better understand the biological roles of critical SARS-CoV-2 proteins, we determined and compared the host transcriptomic responses of the HL-CZ human pro-monocytic cell line upon transfection with key viral genes encoding the spike S1 subunit, S2 subunit, nucleocapsid protein (NP), NSP15 (endoribonuclease), and NSP16 (2'-O-ribose-methyltransferase). RNA sequencing followed by gene set enrichment analysis and other bioinformatics tools revealed that host genes associated with topologically incorrect protein, virus receptor activity, heat shock protein binding, endoplasmic reticulum stress, antigen processing and presentation were up-regulated in the presence of viral spike S1 expression. With spike S2 expression, pro-monocytic genes associated with the interferon-gamma-mediated signaling pathway, regulation of phosphatidylinositol 3-kinase activity, adipocytokine signaling pathway, and insulin signaling pathway were down-regulated, whereas those associated with cytokine-mediated signaling were up-regulated. The expression of NSP15 induced the up-regulation of genes associated with neutrophil degranulation, neutrophil-mediated immunity, oxidative phosphorylation, prion disease, and pathways of neurodegeneration. The expression of NSP16 resulted in the down-regulation of genes associated with S-adenosylmethionine-dependent methyltransferase activity. The expression of NP down-regulated genes associated with positive regulation of neurogenesis, nervous system development, and heart development. Taken together, the complex transcriptomic alterations arising from these viral-host gene interactions offer useful insights into host genes and their pathways that potentially contribute to SARS-CoV-2 pathogenesis.

Project description:BackgroundExperimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties.Methods and findingsHuman mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318-510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one.ConclusionsThe combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.

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SARS Coronavirus Spike Protein Expression in HL-CZ Human Promonocytic Cells: Monoclonal Antibody and Cellular Transcriptomic Analyses

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