visualization-sars-cov-2-using-immuno-rna-fluorescence-situ

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Jagiellonian University

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and  three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed. Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Entry of Human Coronavirus NL63 into the Cell.

The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.

Early events during human coronavirus OC43 entry to the cell.

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization.

Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells.

Virus like particles (VLPs) produced by the expression of viral structural proteins can serve as versatile nanovectors or potential vaccine candidates. In this study we describe for the first time the generation of HCoV-NL63 VLPs using baculovirus system. Major structural proteins of HCoV-NL63 have been expressed in tagged or native form, and their assembly to form VLPs was evaluated. Additionally, a novel procedure for chromatography purification of HCoV-NL63 VLPs was developed. Interestingly, we show that these nanoparticles may deliver cargo and selectively transduce cells expressing the ACE2 protein such as ciliated cells of the respiratory tract. Production of a specific delivery vector is a major challenge for research concerning targeting molecules. The obtained results show that HCoV-NL63 VLPs may be efficiently produced, purified, modified and serve as a delivery platform. This study constitutes an important basis for further development of a promising viral vector displaying narrow tissue tropism.

Novel coronavirus-like particles targeting cells lining the respiratory tract.

Despite high similarity of canine respiratory coronavirus (CRCoV), bovine coronavirus, (BCoV) and human coronavirus OC43 (HCoV-OC43), these viruses differ in species specificity. For years it was believed that they share receptor specificity, utilizing sialic acids for cell surface attachment, internalization, and entry. Interestingly, careful literature analysis shows that viruses indeed bind to the cell surface via sialic acids, but there is no solid data that these moieties mediate virus entry. In our study, using a number of techniques, we showed that all three viruses are indeed able to bind to sialic acids to a different extent, but these molecules render the cells permissive only for the clinical strain of HCoV-OC43, while for others they serve only as attachment receptors. CRCoV and BCoV appear to employ human leukocyte antigen class I (HLA-1) as the entry receptor. Furthermore, we identified heparan sulfate as an alternative attachment factor, but this may be related to the cell culture adaptation, as in ex vivo conditions, it does not seem to play a significant role. Summarizing, we delineated early events during CRCoV, BCoV, and HCoV-OC43 entry and systematically studied the attachment and entry receptor utilized by these viruses.

Canine Respiratory Coronavirus, Bovine Coronavirus, and Human Coronavirus OC43: Receptors and Attachment Factors.

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly,  analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation. It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.

Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor.

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium.

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.

Visualizing Coronavirus Entry into Cells.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen associated with microcephaly and other congenital abnormalities in newborns as well as neurologic complications in adults. The explosive transmission of the virus in the last ten years put it in the limelight and improved our understanding of its biology and pathology. Currently, no vaccine or drugs are available to prevent or treat ZIKV infections. Knowing the potential of flaviviruses to broaden their geographic distribution, as observed for the West Nile virus, it is of importance to develop novel antiviral strategies. In this work, we identified poly(sodium 4-styrenesulfonate) (PSSNa) as a new polymeric ZIKV inhibitor. We demonstrated that PSSNa inhibits ZIKV replication in vitro both in animal and human cells, while no cytotoxicity is observed. Our mechanistic studies indicated that PSSNa acts mostly through direct binding to ZIKV particle and blocking its attachment to the host cells.

In Vitro Inhibition of Zika Virus Replication with Poly(Sodium 4-Styrenesulfonate).

Among seven coronaviruses that infect humans, three (SARS-CoV, MERS-CoV, and the newly identified SARS-CoV-2) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of HCoV-NL63. Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low pathogenic human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using  and  model of human airway epithelium, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry to the host cell by interaction with the S protein. The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of previously developed by us HTCC compound, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses - SARS-CoV-2 and MERS-CoV.

Article	Year
Entry of Human Coronavirus NL63 into the Cell. Journal of virology\| PubMed ID: 29142129	2018
Early events during human coronavirus OC43 entry to the cell. Scientific reports\| PubMed ID: 29740099	2018
Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells. Veterinary research\| PubMed ID: 29970183	2018
Novel coronavirus-like particles targeting cells lining the respiratory tract. PloS one\| PubMed ID: 30183777	2018
Canine Respiratory Coronavirus, Bovine Coronavirus, and Human Coronavirus OC43: Receptors and Attachment Factors. Viruses\| PubMed ID: 30959796	2019
Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor. Journal of virology\| PubMed ID: 31315999	2019
Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium. Journal of virology\| PubMed ID: 32434888	2020
Visualizing Coronavirus Entry into Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32833217	2020
In Vitro Inhibition of Zika Virus Replication with Poly(Sodium 4-Styrenesulfonate). Viruses\| PubMed ID: 32842540	2020
HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV. Journal of virology\| PubMed ID: 33219167	2020

Artur Szczepanski

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