Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2

IntroductionThe spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection.MethodsWe established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice.ResultsAfter infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus.DiscussionThese results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo

Peritoneal Cells Mediate Immune Responses and Cross-Protection Against Influenza A Virus

Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets. However, the responses of peritoneal cells to influenza A virus have not been investigated. Here we show that intraperitoneal inoculation with A/WSN/1933 (H1N1) virus induced virus-reactive IgG production in the peritoneal cavity in mice. The infection resulted in substantial but transient B cell and macrophage depletion along with massive neutrophil infiltration, but virus growth was not detected. Influenza A viruses bound to α-2,6-linked sialic acids of B cells and macrophages and induced apoptotic death of peritoneal cavity cells. However, re-infection with A/WSN/1933 virus did not have adverse effects on immune cells most likely because of the neutralizing antibodies produced in response to the first exposure. Infection of BALB/c mice with A/WSN/1933 induced cross-protection against an otherwise lethal intraperitoneal dose of A/Hongkong/4801/2014 (H3N2) virus. This information suggests that immunological responses in the peritoneal cavity can induce effective defense against future virus infection. Considering the unexpected potent immunoregulatory activity of the peritoneal cells against influenza viruses, we suggest that comparative studies on various immune reactions after infection through different routes may contribute to better selection of vaccination routes in development of efficacious influenza vaccines

Apoptosis Enhances the Replication of Human Coronavirus OC43

Human coronavirus OC43 (HCoV-OC43) is one of the coronaviruses causing a mild common cold, but few studies have been made on this strain. Here, we identified the molecular mechanisms involved in HCoV-OC43-induced apoptosis and its implications for viral reproduction in Vero cells and MRC-5 cells. HCoV-OC43 infection induced apoptosis that was accompanied by cleavage of caspase-3 and PARP, degradation of cyclin D1, and cell cycle arrest at S and G2M phases. Dephosphorylation of STAT1 and STAT3, induced by HCoV-OC43 infection, was also associated with HCoV-OC43-mediated apoptosis. The pan-caspase inhibitor effectively prevented HCoV-OC43-induced apoptosis and reduced viral replication, suggesting that apoptosis contributes to viral replication. Collectively our results indicate that HCoV-OC43 induces caspase-dependent apoptosis to promote viral replication in Vero cells and MRC-5 cells

Apoptosis Enhances the Replication of Human Coronavirus OC43

Human coronavirus OC43 (HCoV-OC43) is one of the coronaviruses causing a mild common cold, but few studies have been made on this strain. Here, we identified the molecular mechanisms involved in HCoV-OC43-induced apoptosis and its implications for viral reproduction in Vero cells and MRC-5 cells. HCoV-OC43 infection induced apoptosis that was accompanied by cleavage of caspase-3 and PARP, degradation of cyclin D1, and cell cycle arrest at S and G2M phases. Dephosphorylation of STAT1 and STAT3, induced by HCoV-OC43 infection, was also associated with HCoV-OC43-mediated apoptosis. The pan-caspase inhibitor effectively prevented HCoV-OC43-induced apoptosis and reduced viral replication, suggesting that apoptosis contributes to viral replication. Collectively our results indicate that HCoV-OC43 induces caspase-dependent apoptosis to promote viral replication in Vero cells and MRC-5 cells

A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model

Mucin1 (MUC1) is a highly glycosylated transmembrane protein that plays a crucial role in the lubrication and protection of normal epithelial cells. However, MUC1 has emerged as a potential target for cancer therapy because it is overexpressed and functions in several types of cancers. Recently, we produced a monoclonal antibody (the anti-hMUC1 antibody) specific to the extracellular region of the MUC1 subunit MUC1-C to evaluate the utility of using anti-MUC1 antibodies in pancreatic cancer models. The anti-hMUC1 antibody recognized the MUC1-C protein in pancreatic cancer cells. Based on immunostaining and confocal image analyses, the anti-hMUC1 antibody initially bound to the cell membrane then was internalized in cancer cells that express MUC1. The anti-hMUC1 antibody suppressed epidermal growth factor (EGF)-mediated extracellular signal–regulated kinase (ERK) phosphorylation and cyclin D1 expression. When the anti-hMUC1 antibody was injected into a xenograft mouse model and traced using an in vivo imaging system, we observed that the anti-hMUC1 antibody was localized to MUC1-expressing pancreatic tumors. Importantly, the anti-hMUC1 monoclonal antibody suppressed pancreatic tumor growth in mice. According to immunohistochemistry analysis using a pancreatic cancer tissue array and the anti-hMUC1 antibody, MUC1 was highly expressed in human pancreatic cancer tissues compared to normal tissues. Therefore, we conclude that the anti-hMUC1 antibody specifically targets MUC1 and suppresses its function in pancreatic cancer in vitro and in vivo and can be further developed as a promising targeted therapy to treat pancreatic cancer

Rk1, a Ginsenoside, Is a New Blocker of Vascular Leakage Acting through Actin Structure Remodeling

<div><p>Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage.</p> </div

Rk1, but not Rg1 or Rb1, inhibits VEGF-induced retinal endothelial permeability.

<p>(<b>A</b>) HRECs were plated onto a Transwellfilter. After reaching confluence, HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. (<b>B</b>) The structure of gensenosides. (<b>C</b>) HRECs were plated onto a Transwell filter. After reaching confluence, HRECs were pretreated for 40 min with or without Rb1, Rg1, and Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. All data are presented as the mean ± S.E. from three different experiments performed in duplicate. **, <i>P</i> < 0.01 <i>vs</i>. VEGF alone.</p

AGE induces retinal endothelial permeability <i>via</i> expression of VEGF.

<p>(<b>A</b>, <b>B</b>) HRECs were stimulated with 5 µM AGE-BSA for the indicated times. Total mRNAs were isolated and RT-PCR was performed with specific primers for human VEGF. GAPDH served as an internal control. (B) Ratio of VEGF mRNA to GAPDH mRNA. (<b>C</b>) HRECs were plated onto a Transwell filter. After reaching confluence, HRECs were pretreated for 30 min with or without 1C11 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. Data are the mean ± S.E. **, <i>p</i> < 0.01 <i>vs</i>. AGE alone. (<b>D</b>) Confluent HRECs were incubated for 30 min with or without 10 µg/ml 1C11 and stimulated with 5 µM AGE-BSA for 10 h. Translocations of TJ proteins were assessed, as described in “Experimental Procedures”. The Triton X-100-insoluble and soluble fractions were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with anti-ZO-1, anti-ZO-2, and anti-occludin antibodies. Blots are representative of three independent experiments. (<b>E</b>) Densitometric analyses are presented as the relative ratio of tight junction proteins in membrane and cytosol and quantified with NIH ImageJ software. Data are the mean ± S.E. **, <i>p</i> < 0.01 <i>vs</i>. control. ^##, <i>p</i> < 0.01 <i>vs</i>. AGE alone.</p

Rk1 induces cortactin phosphorylation.

<p>(<b>A</b>, <b>B</b>) Confluent HRECs were treated with Rk1 (0.1, 1, 5, or 10 µg/ml) for the indicated times. Cell lysates were subject to affinity precipitation using anti-cortactin antibody. Total cortactin indicates total amount of active and inactive cortactin in the HRECs. Western blot was performed using an antibody specific for phospho-tyrosine (PY) and cortactin (Cort). (<b>C</b>) Confluent HRECs were treated with Rk1 (10 µg/ml) for 30 min. The cells were then fixed and stained with Rhodamine Phalloidin and anti-cortactin antibody. Arrows indicate cortactin. (<b>D</b>) Confluent HRECs were pretreated for 1 h with or without VEGF (20 ng/ml) prior to stimulation with Rk1 (0.5, 1, 5, or 10 µg/ml) for 1 h. The cells were then fixed and processed for immunofluorescence staining using Rhodamine Phalloidin. The samples were viewed by fluorescence microscopy.</p