Inhibition of SARS-CoV-2 infection in cellular systems using engineered trimeric receptor-binding domain of spike protein.

Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest. For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1.

Trimeric receptor-binding domain of SARS-CoV-2 acts as a potent inhibitor of ACE2 receptor-mediated viral entry.

The COVID-19 pandemic has caused over four million deaths and effective methods to control CoV-2 infection, in addition to vaccines, are needed. The CoV-2 binds to the ACE2 on human cells through the receptor-binding domain (RBD) of the trimeric spike protein. Our modeling studies show that a modified trimeric RBD (tRBD) can interact with three ACE2 receptors, unlike the native spike protein, which binds to only one ACE2. We found that tRBD binds to the ACE2 with 58-fold higher affinity than monomeric RBD (mRBD) and blocks spike-dependent pseudoviral infection over 4-fold more effectively compared to the mRBD. Although mRBD failed to block CoV-2 USA-WA1/2020 infection, tRBD efficiently blocked the true virus infection in plaque assays. We show that tRBD is a potent inhibitor of CoV-2 through both competitive binding to the ACE2 and steric hindrance, and has the potential to emerge as a first-line therapeutic method to control COVID-19.

HBD-2 binds SARS-CoV-2 RBD and blocks viral entry: Strategy to combat COVID-19.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19-related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell-derived host defense peptide that has anti-viral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor-binding domain (RBD) (KD ∼ 2µM by surface plasmon resonance), preventing it from binding to ACE2-expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG-mediated infection, of ACE2-expressing human cells with an IC50 of 2.8 ± 0.4 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.

Sam68 contributes to intestinal inflammation in experimental and human colitis.

Sam68 is an RNA-binding protein with an adaptor role in signal transduction. Our previous work identified critical proinflammatory and apoptotic functions for Sam68, downstream of the TNF/TNFR1 and TLR2/3/4 pathways. Recent studies have shown elevated Sam68 in inflamed tissues from rheumatoid arthritis and ulcerative colitis (UC) patients, suggesting that Sam68 contributes to chronic inflammatory diseases. Here, we hypothesized that deletion of Sam68 is protective against experimental colitis in vivo, via reductions in TNF-associated inflammatory signaling. We used Sam68 knockout (KO) mice to study the role of Sam68 in experimental colitis, including its contributions to TNF-induced inflammatory gene expression in three-dimensional intestinal organoid cultures. We also studied the expression of Sam68 and inflammatory genes in colon tissues of UC patients. Sam68 KO mice treated with an acute course of DSS exhibited significantly less weight loss and histopathological inflammation compared to wild-type controls, suggesting that Sam68 contributes to experimental colitis. Bone marrow transplants showed no pathologic role for hematopoietic cell-specific Sam68, suggesting that non-hematopoietic Sam68 drives intestinal inflammation. Gene expression analyses showed that Sam68 deficiency reduced the expression of proinflammatory genes in colon tissues from DSS-treated mice, as well as TNF-treated three-dimensional colonic organoids. We also found that inflammatory genes, such as TNF, CCR2, CSF2, IL33 and CXCL10, as well as Sam68 protein, were upregulated in inflamed colon tissues of UC patients. This report identifies Sam68 as an important inflammatory driver in response to intestinal epithelial damage, suggesting that targeting Sam68 may hold promise to treat UC patients.

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ~ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4± 0.1 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

Regulation of B-cell function by NF-kappaB c-Rel in health and disease.

B cells mediate humoral immune response and contribute to the regulation of cellular immune response. Members of the Nuclear Factor kappaB (NF-κB) family of transcription factors play a major role in regulating B-cell functions. NF-κB subunit c-Rel is predominantly expressed in lymphocytes, and in B cells, it is required for survival, proliferation, and antibody production. Dysregulation of c-Rel expression and activation alters B-cell homeostasis and is associated with B-cell lymphomas and autoimmune pathologies. Based on its essential roles, c-Rel may serve as a potential prognostic and therapeutic target. This review summarizes the current understanding of the multifaceted role of c-Rel in B cells and B-cell diseases.