Biology subjects

Lew, J.

Publications and source records attributed to Lew, J..

6 recordsLinked to original sources

bioRxiv2025

Genome-wide Screening Identifies Unique Host-Directed Drugs and Pro-viral Signalling Pathways for SARS-CoV-2

SARS-CoV-2 is a positive-sense RNA virus and was responsible for the devastating COVID-19 pandemic. Although the current disease burden is less severe, there are limited treatment options, significant gaps in knowledge, and a looming threat of the emergence of variants and future pandemics. To address these challenges, we performed genome-wide CRISPR knockout screens in a novel human lung cell line NCI-H23ACE2, as well as in HEK293TACE2 cells, with SARS-CoV-2 Wuhan virus, with the aim of identifying host-dependency factors that could predict effective antivirals. We identified four host-directed drugs, donepezil, dH-ergocristine, trametinib and sorafenib, that could potentially be repurposed to treat coronavirus infections. Three of the drugs inhibited SARS-CoV-2, HCoV-229E, and HCoV-OC43, suggesting they could be used as pan-coronavirus antivirals. We also confirmed that SARS-CoV-2 relies on the NRAS/Raf/MEK/ERK signaling pathway for its replication. Our study highlights the robustness and efficiency of a bilateral approach of gene silencing and antiviral screening to identify host-dependency factors and effective antivirals.

microbiology↗

bioRxiv2023

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) in thyrocytes induces follicular hyperplasia and impairs thyroid hormone synthesis

The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathways overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. To elucidate mTORC1s role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting Tuberous Sclerosis Complex 2 (TSC2), an intracellular inhibitor of mTORC1. The resulting TPO-TSC2KO mice exhibited a significant reduction in TSC2 levels, leading to a six-fold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, accompanied by heterogeneity among thyroid follicles, larger follicles, increased colloid and epithelium. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated Cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone (TH) biosynthesis, including thyroglobulin, thyroid peroxidase, and sodium-iodide symporter (NIS), while TTF1, PAX8, and MCT8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although T4, T3 and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared to littermate control mice. TPO-TSC2KO mice aged to 12 months or older developed aberrant thyroid conditions, including follicular hyperplasia, inflammation, and thyroid tumors. In conclusion, our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits TH biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation. Chronic mTORC1 activation leads to thyroid tumor formation, highlighting the role of mTORC1 in thyroid dysfunction and tumorigenesis.

physiology↗

bioRxiv2023

Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology

The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology and titers were comparable between rescued molecular clones and a clinical isolate (VIDO-01 strain), thus providing confidence that the rescued viruses can be used as effective replication tools. Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells, were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death and a novel lung cell line NCI-H23ACE2 A3 showed about [~]99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection and are currently being used in CRISPR knockout and activation screens in our lab. ImportanceWe used a reverse genetics system to generate a wild type as well as a nanoluciferase-expressing reporter clone of SARS-CoV-2. The reporter virus allows for rapid transient replication assays and high throughput screens by detection of virus replication using luciferase assays. In addition, the reverse genetic system can be used to generate mutant viruses to study phenotypes of variant mutations. Additionally, unique human lung cell lines supporting SARS-CoV-2 replication will aid in studying the virus in a lung-relevant environment and based on high cytopathology induced in some cell lines, will be useful for screens that rely on virus-induced cell death for selection. Our study aims to enhance and contribute to the current replication tools available to study SARS-CoV-2 by providing rapid methods, virus clones and novel lung cell lines.

molecular biology↗

bioRxiv2022

An S1 subunit vaccine and combination adjuvant (COVAC-1) elicits robust protection against SARS-CoV-2 challenge in African green monkeys

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the ongoing global pandemic. With over 500 million cases and more than 6 million deaths reported globally, the need for access to effective vaccines is clear. An ideal SARS-CoV-2 vaccine will prevent pathology in the lungs and prevent virus replication in the upper respiratory tract, thus reducing transmission. Here, we assessed the efficacy of an adjuvanted SARS-CoV-2 S1 subunit vaccine, called COVAC-1, in an African green monkey (AGM) model. AGMs immunized and boosted with COVAC-1 were protected from SARS-CoV-2 challenge compared to unvaccinated controls based on reduced pathology and reduced viral RNA levels and infectious virus in the respiratory tract. Both neutralizing antibodies and antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) were observed in vaccinated animals prior to the challenge. COVAC-1 induced effective protection, including in the upper respiratory tract, thus supporting further development and utility for determining the mechanism that confers this protection. AUTHOR SUMMARYVaccines that can prevent the onward transmission of SARS-CoV-2 and prevent disease are highly desirable. Whether this can be accomplished without mucosal immunization by a parenterally administered subunit vaccine is not well established. Here we demonstrate that following two vaccinations, a protein subunit vaccine containing the S1 portion of the SARS-CoV-2 spike glycoprotein and the novel adjuvant TriAdj significantly reduces the amount of virus in the lungs and also mediates rapid clearance of the virus from the upper respiratory tract. Further support of the effectiveness of COVAC-1 was the observation of reduced pathology in the lungs and viral RNA being largely absent from tissues, blood, and rectal swabs. Thus COVAC-1 appears promising at mediating protection in both the upper and lower respiratory tract and may be capable of reducing subsequent transmission of SARS-CoV-2. Further investigation into the mechanism of protection in the upper respiratory tract and the initial immune response that supports this would be warranted.

microbiology↗

bioRxiv2022

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies, and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

immunology↗

bioRxiv2021

Male sex and age biases viral burden, viral shedding, and type 1 and 2 interferon responses during SARS-CoV-2 infection in ferrets

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and the elderly. Here we investigated the impact of male sex and age by infecting adult male, aged male, and adult female ferrets with SARS-CoV-2. Aged male ferrets had a decrease in temperature which was accompanied by prolonged viral replication with increased pathology in the upper respiratory tract after infection. Transcriptome analysis of the nasal turbinates and lungs indicated that female ferrets had significant increases in interferon response genes (OASL, MX1, ISG15, etc.) on day 2 post infection which was delayed in aged males. In addition, genes associated with taste and smell such as RTP1, CHGA, and CHGA1 at later time points were upregulated in males but not in females. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

microbiology↗