Comparative assessment of favipiravir and remdesivir against human coronavirus NL63 in molecular docking and cell culture models.

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.

Viral polymerase binding and broad-spectrum antiviral activity of molnupiravir against human seasonal coronaviruses.

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.

Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses.

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks.

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63.

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as ß-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC50 = 0.64 µg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC50 values against virus yield were 1.52 µM and 2.60 µM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC50 = 0.06 µM) and indigodole B (IC50 = 2.09 µM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

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

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, in silico 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.IMPORTANCE 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.

Entry of Human Coronavirus NL63 into the Cell.

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 ex vivo 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.IMPORTANCE 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.

Structural Characterization of Human Coronavirus NL63 N Protein.

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.

Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity.

A series of doubly flexible nucleoside analogues were designed based on the acyclic sugar scaffold of acyclovir and the flex-base moiety found in the fleximers. The target compounds were evaluated for their antiviral potential and found to inhibit several coronaviruses. Significantly, compound 2 displayed selective antiviral activity (CC50 >3× EC50) towards human coronavirus (HCoV)-NL63 and Middle East respiratory syndrome-coronavirus, but not severe acute respiratory syndrome-coronavirus. In the case of HCoV-NL63 the activity was highly promising with an EC50 <10 µM and a CC50 >100 µM. As such, these doubly flexible nucleoside analogues are viewed as a novel new class of drug candidates with potential for potent inhibition of coronaviruses.

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells.

UNLABELLED: Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE: ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

Stability of infectious human coronavirus NL63.

The human coronavirus NL63 was identified in 2004 and subsequent studies showed its worldwide distribution. Infection with this pathogen is associated with upper and lower respiratory tract diseases of mild to moderate severity. Furthermore, HCoV-NL63 is the main cause of croup in children. Within this study an optimal protocol for freeze-drying that allows safe and effective preservation of HCoV-NL63 infectious material was developed. Lyophilized virus preparations can be stored either at ambient temperature or at +4°C. In the latter case samples may be stored for at least two months. Surprisingly, conducted analysis showed that HCoV-NL63 virions are exquisitely stable in liquid media and can be stored also without preservatives at ambient temperature for up to 14 days.

Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir.

Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Müller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schöpf, J., Kögl, M., Friedel, C., Müller, M.A., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Züst, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Weßels, C., Pöhlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.

Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells.

BACKGROUND: Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts. METHODS: Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing. RESULTS: Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed. CONCLUSIONS: At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.

Novel polymeric inhibitors of HCoV-NL63.

The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Replication-dependent downregulation of cellular angiotensin-converting enzyme 2 protein expression by human coronavirus NL63.

Like severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus (HCoV)-NL63 employs angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. SARS-CoV infection causes robust downregulation of cellular ACE2 expression levels and it has been suggested that the SARS-CoV effect on ACE2 is involved in the severity of disease. We investigated whether cellular ACE2 downregulation occurs at optimal replication conditions of HCoV-NL63 infection. The expression of the homologue of ACE2, the ACE protein not used as a receptor by HCoV-NL63, was measured as a control. A specific decrease for ACE2 protein level was observed when HCoV-NL63 was cultured at 34 °C. Culturing the virus at the suboptimal temperature of 37 °C resulted in low replication of the virus and the effect on ACE2 expression was lost. We conclude that the decline of ACE2 expression is dependent on the efficiency of HCoV-NL63 replication, and that HCoV-NL63 and SARS-CoV both affect cellular ACE2 expression during infection.

A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.

For antiviral screenings purposes, infection of cell cultures with the virus under study, should ideally result in the induction, within just a few days, of (nearly) complete CPE and allow the calculation of acceptable Z' factors (>0.5). The human Corona virus NL63 (HCoV-NL63) causes only limited CPE on different cell lines (Schildgen et al., 2006). Following infection of Vero118 cells, virus-induced CPE was too low to allow readout based on classical colorimetric methods (such as the MTS assay), even following prolonged incubation times (>7 days). To develop an antiviral screenings-assay against HCoV-NL63, we explored whether a dead-cell protease substrate could be used instead. The substrate used is a quenched peptide (bis-AAF-R110) that releases a fluorophore upon proteolytic-cleavage by proteases; the latter released from dead cells. Following different rounds of optimization a screening protocol was developed: Vero118 cells in 96-well plate format were infected with HCoV-NL63 (MOI=0.01; 200µL cell culture; 2.10(4)cells/mL, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35°C after which 20µL of the peptide solution was added. Fluorescence was quantitated 2 hr after incubation at 37°C. A roughly 3-fold increase in fluorescence intensity in the infected cultures was observed as compared to the uninfected cultures with a low well-to-well variability. Z' factors calculated from different experiments were in the range of 0.6-0.8, indicating excellent assay quality. An anti-ACE2 polyclonal antiserum (that prevents coronavirus infection in cell cultures) was used as a positive control and allowed to validate the assay for antiviral screening purposes. In conclusion, in conditions where a viability staining is inadequate to quantitate virus-induced CPE, a novel simple and convenient method that detects cell-death and that is suitable for high-throughput screening purposes can be employed.

Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506.

Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry.

The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.