P21-activated kinase 1 (PAK1)-mediated cytoskeleton rearrangement promotes SARS-CoV-2 entry and ACE2 autophagic degradation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has had a significant impact on healthcare systems and economies worldwide. The continuous emergence of new viral strains presents a major challenge in the development of effective antiviral agents. Strategies that possess broad-spectrum antiviral activities are desirable to control SARS-CoV-2 infection. ACE2, an angiotensin-containing enzyme that prevents the overactivation of the renin angiotensin system, is the receptor for SARS-CoV-2. ACE2 interacts with the spike protein and facilitates viral attachment and entry into host cells. Yet, SARS-CoV-2 infection also promotes ACE2 degradation. Whether restoring ACE2 surface expression has an impact on SARS-CoV-2 infection is yet to be determined. Here, we show that the ACE2-spike complex is endocytosed and degraded via autophagy in a manner that depends on clathrin-mediated endocytosis and PAK1-mediated cytoskeleton rearrangement. In contrast, free cellular spike protein is selectively cleaved into S1 and S2 subunits in a lysosomal-dependent manner. Importantly, we show that the pan-PAK inhibitor FRAX-486 restores ACE2 surface expression and suppresses infection by different SARS-CoV-2 strains. FRAX-486-treated Syrian hamsters exhibit significantly decreased lung viral load and alleviated pulmonary inflammation compared with untreated hamsters. In summary, our findings have identified novel pathways regulating viral entry, as well as therapeutic targets and candidate compounds for controlling the emerging strains of SARS-CoV-2 infection.

SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo.

"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

The spike receptor-binding motif G496S substitution determines the replication fitness of SARS-CoV-2 Omicron sublineage.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Phosphatidic acid phosphatase 1 impairs SARS-CoV-2 replication by affecting the glycerophospholipid metabolism pathway.

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.

Pathogenicity, transmissibility, and fitness of SARS-CoV-2 Omicron in Syrian hamsters.

The in vivo pathogenicity, transmissibility, and fitness of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant are not well understood. We compared these virological attributes of this new variant of concern (VOC) with those of the Delta (B.1.617.2) variant in a Syrian hamster model of COVID-19. Omicron-infected hamsters lost significantly less body weight and exhibited reduced clinical scores, respiratory tract viral burdens, cytokine and chemokine dysregulation, and lung damage than Delta-infected hamsters. Both variants were highly transmissible through contact transmission. In noncontact transmission studies Omicron demonstrated similar or higher transmissibility than Delta. Delta outcompeted Omicron without selection pressure, but this scenario changed once immune selection pressure with neutralizing antibodies-active against Delta but poorly active against Omicron-was introduced. Next-generation vaccines and antivirals effective against this new VOC are therefore urgently needed.

SARS-CoV-2 infection induces inflammatory bone loss in golden Syrian hamsters.

Extrapulmonary complications of different organ systems have been increasingly recognized in patients with severe or chronic Coronavirus Disease 2019 (COVID-19). However, limited information on the skeletal complications of COVID-19 is known, even though inflammatory diseases of the respiratory tract have been known to perturb bone metabolism and cause pathological bone loss. In this study, we characterize the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on bone metabolism in an established golden Syrian hamster model for COVID-19. SARS-CoV-2 causes significant multifocal loss of bone trabeculae in the long bones and lumbar vertebrae of all infected hamsters. Moreover, we show that the bone loss is associated with SARS-CoV-2-induced cytokine dysregulation, as the circulating pro-inflammatory cytokines not only upregulate osteoclastic differentiation in bone tissues, but also trigger an amplified pro-inflammatory cascade in the skeletal tissues to augment their pro-osteoclastogenesis effect. Our findings suggest that pathological bone loss may be a neglected complication which warrants more extensive investigations during the long-term follow-up of COVID-19 patients. The benefits of potential prophylactic and therapeutic interventions against pathological bone loss should be further evaluated.

Probable Animal-to-Human Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant AY.127 Causing a Pet Shop-Related Coronavirus Disease 2019 (COVID-19) Outbreak in Hong Kong.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human and other mammals, including hamsters. Syrian (Mesocricetus auratus) and dwarf (Phodopus sp.) hamsters are susceptible to SARS-CoV-2 infection in the laboratory setting. However, pet shop-related Coronavirus Disease 2019 (COVID-19) outbreaks have not been reported. METHODS: We conducted an investigation of a pet shop-related COVID-19 outbreak due to Delta variant AY.127 involving at least 3 patients in Hong Kong. We tested samples collected from the patients, environment, and hamsters linked to this outbreak and performed whole genome sequencing analysis of the reverse transcription polymerase chain reaction (RT-PCR)-positive samples. RESULTS: The patients included a pet shop keeper (Patient 1), a female customer of the pet shop (Patient 2), and the husband of Patient 2 (Patient 3). Investigation showed that 17.2% (5/29) and 25.5% (13/51) environmental specimens collected from the pet shop and its related warehouse, respectively, tested positive for SARS-CoV-2 RNA by RT-PCR. Among euthanized hamsters randomly collected from the storehouse, 3% (3/100) tested positive for SARS-CoV-2 RNA by RT-PCR and seropositive for anti-SARS-CoV-2 antibody by enzyme immunoassay. Whole genome analysis showed that although all genomes from the outbreak belonged to the Delta variant AY.127, there were at least 3 nucleotide differences among the genomes from different patients and the hamster cages. Genomic analysis suggests that multiple strains have emerged within the hamster population, and these different strains have likely transmitted to human either via direct contact or via the environment. CONCLUSIONS: Our study demonstrated probable hamster-to-human transmission of SARS-CoV-2. As pet trading is common around the world, this can represent a route of international spread of this pandemic virus.

hnRNP C modulates MERS-CoV and SARS-CoV-2 replication by governing the expression of a subset of circRNAs and cognitive mRNAs.

ABSTRACTHost circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA-mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.

Low Environmental Temperature Exacerbates Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Golden Syrian Hamsters.

BACKGROUND: The effect of low environmental temperature on viral shedding and disease severity of Coronavirus Disease 2019 (COVID-19) is uncertain. METHODS: We investigated the virological, clinical, pathological, and immunological changes in hamsters housed at room (21°C), low (12-15°C), and high (30-33°C) temperature after challenge by 105 plaque-forming units of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RESULTS: The nasal turbinate, trachea, and lung viral load and live virus titer were significantly higher (~0.5-log10 gene copies/ß-actin, P < .05) in the low-temperature group at 7 days postinfection (dpi). The low-temperature group also demonstrated significantly higher level of tumor necrosis factor-α, interferon-γ (IFN-γ), interleukin-1ß, and C-C motif chemokine ligand 3, and lower level of the antiviral IFN-α in lung tissues at 4 dpi than the other 2 groups. Their lungs were grossly and diffusely hemorrhagic, with more severe and diffuse alveolar and peribronchiolar inflammatory infiltration, bronchial epithelial cell death, and significantly higher mean total lung histology scores. By 7 dpi, the low-temperature group still showed persistent and severe alveolar inflammation and hemorrhage, and little alveolar cell proliferative changes of recovery. The viral loads in the oral swabs of the low-temperature group were significantly higher than those of the other two groups from 10 to 17 dpi by about 0.5-1.0 log10 gene copies/ß-actin. The mean neutralizing antibody titer of the low-temperature group was significantly (P < .05) lower than that of the room temperature group at 7 dpi and 30 dpi. CONCLUSIONS: This study provided in vivo evidence that low environmental temperature exacerbated the degree of virus shedding, disease severity, and tissue proinflammatory cytokines/chemokines expression, and suppressed the neutralizing antibody response of SARS-CoV-2-infected hamsters. Keeping warm in winter may reduce the severity of COVID-19.

Severe fever with thrombocytopenia syndrome virus (SFTSV)-host interactome screen identifies viral nucleoprotein-associated host factors as potential antiviral targets.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that causes severe infection in humans characterized by an acute febrile illness with thrombocytopenia and hemorrhagic complications, and a mortality rate of up to 30%. Understanding on virus-host protein interactions may facilitate the identification of druggable antiviral targets. Herein, we utilized liquid chromatography-tandem mass spectrometry to characterize the SFTSV interactome in human embryonic kidney-derived permanent culture (HEK-293T) cells. We identified 445 host proteins that co-precipitated with the viral glycoprotein N, glycoprotein C, nucleoprotein, or nonstructural protein. A network of SFTSV-host protein interactions based on reduced viral fitness affected upon host factor down-regulation was then generated. Screening of the DrugBank database revealed numerous drug compounds that inhibited the prioritized host factors in this SFTSV interactome. Among these drug compounds, the clinically approved artenimol (an antimalarial) and omacetaxine mepesuccinate (a cephalotaxine) were found to exhibit anti-SFTSV activity in vitro. The higher selectivity of artenimol (71.83) than omacetaxine mepesuccinate (8.00) highlights artenimol's potential for further antiviral development. Mechanistic evaluation showed that artenimol interfered with the interaction between the SFTSV nucleoprotein and the host glucose-6-phosphate isomerase (GPI), and that omacetaxine mepesuccinate interfered with the interaction between the viral nucleoprotein with the host ribosomal protein L3 (RPL3). In summary, the novel interactomic data in this study revealed the virus-host protein interactions in SFTSV infection and facilitated the discovery of potential anti-SFTSV treatments.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that poses significant threats to global public health. Macrophages and dendritic cells are both key sentinel cells in the host immune response and play critical roles in the pathogenesis of flavivirus infections. Recent studies showed that ZIKV could productively infect monocyte-derived dendritic cells (moDCs), but the role of macrophages in ZIKV infection remains incompletely understood. In this study, we first compared ZIKV infection in monocyte-derived macrophages (MDMs) and moDCs derived from the same donors. We demonstrated that while both MDMs and moDCs were susceptible to epidemic (Puerto Rico) and pre-epidemic (Uganda) strains of ZIKV, virus replication was largely restricted in MDMs but not in moDCs. ZIKV induced significant apoptosis in moDCs but not MDMs. The restricted virus replication in MDMs was not due to inefficient virus entry but was related to post-entry events in the viral replication cycle. In stark contrast with moDCs, ZIKV failed to inhibit STAT1 and STAT2 phosphorylation in MDMs. This resulted in the lack of efficient antagonism of the host type I interferon-mediated antiviral responses. Importantly, depletion of STAT2 but not STAT1 in MDMs significantly rescued the replication of ZIKV and the prototype flavivirus yellow fever virus. Overall, our findings revealed a differential interplay between macrophages and dendritic cells with ZIKV. While dendritic cells may be exploited by ZIKV to facilitate virus replication, macrophages restricted ZIKV infection.

In silico structure-based discovery of a SARS-CoV-2 main protease inhibitor.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (Mpro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 Mpro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 Mpro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.

Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007-0.35 µg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1-3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine.

Metallodrug ranitidine bismuth citrate suppresses SARS-CoV-2 replication and relieves virus-associated pneumonia in Syrian hamsters.

SARS-CoV-2 is causing a pandemic of COVID-19, with high infectivity and significant mortality1. Currently, therapeutic options for COVID-19 are limited. Historically, metal compounds have found use as antimicrobial agents, but their antiviral activities have rarely been explored. Here, we test a set of metallodrugs and related compounds, and identify ranitidine bismuth citrate, a commonly used drug for the treatment of Helicobacter pylori infection, as a potent anti-SARS-CoV-2 agent, both in vitro and in vivo. Ranitidine bismuth citrate exhibited low cytotoxicity and protected SARS-CoV-2-infected cells with a high selectivity index of 975. Importantly, ranitidine bismuth citrate suppressed SARS-CoV-2 replication, leading to decreased viral loads in both upper and lower respiratory tracts, and relieved virus-associated pneumonia in a golden Syrian hamster model. In vitro studies showed that ranitidine bismuth citrate and its related compounds exhibited inhibition towards both the ATPase (IC50 = 0.69 µM) and DNA-unwinding (IC50 = 0.70 µM) activities of the SARS-CoV-2 helicase via an irreversible displacement of zinc(II) ions from the enzyme by bismuth(III) ions. Our findings highlight viral helicase as a druggable target and the clinical potential of bismuth(III) drugs or other metallodrugs for the treatment of SARS-CoV-2 infection.