Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

[3-O-ß-chacotriosyl glycyrrhetinic acid derivatives as potential small-molecule SARS-CoV-2 fusion inhibitors against SARS-CoV-2 entry into host cells].

OBJECTIVE: To study the inhibitory activities of 3-O-ß-chacotriosyl glycyrrhetinic acid derivatives against the entry of SARS-CoV-2 into host cells. METHODS: With pentacyclic triterpene saponin glycyrrhizic acid (a natural SARS-CoV-2 entry inhibitor) as the lead compound, a series of 3-O-ß-chacotriosyl glycyrrhetinic acid derivatives were designed and synthesized based on hypridization principle, and their inhibitory activities against virus entry were tested in SARS-CoV-2 pseudovirusinfected cells. The antiviral targets of the lead compound 1b was identified by pseudotyped SARS-CoV-2 infection assay and surface plasmon resonance (SPR) assay, and the S protein-mediated cell-cell fusion assay was used to evaluate the effect of 1b on virus-cell membrane fusion. Molecular docking and single amino acid mutagenesis were carried out to analyze the effect of 1b on binding activitiy of S protein. RESULTS: The lead compound 1b showed significant inhibitory effect against Omicron pseudovirus with an EC50 value of 3.28 µmol/L (P < 0.05), and had broad-spectrum antiviral activity against other SARS-CoV-2 pseudovirus. Spike-dependent cell-cell fusion assay demonstrated an inhibitory effect of 1b against SARS-CoV-2 S proteinmediated cell-cell fusion. Molecular docking analysis predicted that the lead compound 1b could be well fitted into a cavity between the attachment (S1) and fusion (S2) subunits at the 3-fold axis, where it formed multiple hydrophobic interactions with Glu309, Ser305, Arg765 and Lys964 residues with a KD value of -8.6 kcal/mol. The compound 1b at 10, 5, 2.5 and 1.25 µmol/L showed a significantly reduced inhibitory activity against the pseudovirus with mutated Arg765, Lys964, Glu309 and Leu303 (P < 0.01). CONCLUSION: 3-O-ß-chacotriosyl glycyrrhetinic acid derivatives are capable of stabilizing spike protein in the pre-fusion step to interfere with the fusion of SARS-CoV-2 with host cell membrane, and can thus serve as potential novel small-molecule SARS-CoV-2 fusion inhibitors.

SARS-CoV-2 infects cells lining the blood-retinal barrier and induces a hyperinflammatory immune response in the retina via systemic exposure.

SARS-CoV-2 has been shown to cause wide-ranging ocular abnormalities and vision impairment in COVID-19 patients. However, there is limited understanding of SARS-CoV-2 in ocular transmission, tropism, and associated pathologies. The presence of viral RNA in corneal/conjunctival tissue and tears, along with the evidence of viral entry receptors on the ocular surface, has led to speculation that the eye may serve as a potential route of SARS-CoV-2 transmission. Here, we investigated the interaction of SARS-CoV-2 with cells lining the blood-retinal barrier (BRB) and the role of the eye in its transmission and tropism. The results from our study suggest that SARS-CoV-2 ocular exposure does not cause lung infection and moribund illness in K18-hACE2 mice despite the extended presence of viral remnants in various ocular tissues. In contrast, intranasal exposure not only resulted in SARS-CoV-2 spike (S) protein presence in different ocular tissues but also induces a hyperinflammatory immune response in the retina. Additionally, the long-term exposure to viral S-protein caused microaneurysm, retinal pigmented epithelium (RPE) mottling, retinal atrophy, and vein occlusion in mouse eyes. Notably, cells lining the BRB, the outer barrier, RPE, and the inner barrier, retinal vascular endothelium, were highly permissive to SARS-CoV-2 replication. Unexpectedly, primary human corneal epithelial cells were comparatively resistant to SARS-CoV-2 infection. The cells lining the BRB showed induced expression of viral entry receptors and increased susceptibility towards SARS-CoV-2-induced cell death. Furthermore, hyperglycemic conditions enhanced the viral entry receptor expression, infectivity, and susceptibility of SARS-CoV-2-induced cell death in the BRB cells, confirming the reported heightened pathological manifestations in comorbid populations. Collectively, our study provides the first evidence of SARS-CoV-2 ocular tropism via cells lining the BRB and that the virus can infect the retina via systemic permeation and induce retinal inflammation.

Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Differentiating Cell Entry Potentials of SARS-CoV-2 Omicron Subvariants on Human Lung Epithelium Cells.

The surface spike (S) glycoprotein mediates cell entry of SARS-CoV-2 into the host through fusion at the plasma membrane or endocytosis. Omicron lineages/sublineages have acquired extensive mutations in S to gain transmissibility advantages and altered antigenicity. The fusogenicity, antigenicity, and evasion of Omicron subvariants have been extensively investigated at unprecedented speed to align with the mutation rate of S. Cells that overexpress receptors/cofactors are mostly used as hosts to amplify infection sensitivity to tested variants. However, systematic cell entry comparisons of most prior dominant Omicron subvariants using human lung epithelium cells are yet to be well-studied. Here, with human bronchial epithelium BEAS-2B cells as the host, we compared single-round virus-to-cell entry and cell-to-cell fusion of Omicron BA.1, BA.5, BQ.1.1, CH.1.1, XBB.1.5, and XBB.1.16 based upon split NanoLuc fusion readout assays and the S-pseudotyped lentivirus system. Virus-to-cell entry of tested S variants exhibited cell-type dependence. The parental Omicron BA.1 required more time to develop full entry to HEK293T-ACE2-TMPRSS2 than BEAS-2B cells. Compared to unchanged P681, S-cleavage constructs of P681H/R did not have any noticeable advantages in cell entry. Omicron BA.1 and its descendants entered BEAS-2B cells more efficiently than D614G, and it was slightly less or comparable to that of Delta. Serine protease-pretreated Omicron subvariants enhanced virus-to-cell entry in a dose-dependent manner, suggesting fusion at the plasma membrane persists as a productive cell entry route. Spike-mediated cell-to-cell fusion and total S1/S2 processing of Omicron descendants were similar. Our results indicate no obvious entry or fusion advantages of recent Omicron descendants over preceding variants since Delta, thus supporting immune evasion conferred by antigenicity shifts due to altered S sequences as probably the primary viral fitness driver.

Inhibition of PEDV viral entry upon blocking N-glycan elaboration.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the global swine industry, demanding a thorough understanding of its cellular invasion mechanism for effective interventions. This study meticulously investigates the impact of O- and N-linked glycans on PEDV proteins and host cell interaction, shedding light on their influence on the virus's invasion process. Utilizing CRISPR-Cas9 technology to inhibit cell surface O- and N-linked glycan synthesis demonstrated no discernible impact on virus infection. However, progeny PEDV strains lacking these glycans exhibited a minor effect of O-linked glycans on virus infection. Conversely, a notable 40% reduction in infectivity was observed when the virus surface lacked N-linked glycans, emphasizing their pivotal role in facilitating virus recognition and binding to host cells. Additionally, inhibition studies utilizing kifunensine, a natural glycosidase I inhibitor, reaffirmed the significant role of N-linked glycans in virus infection. Inhibiting N-linked glycan synthesis with kifunensine substantially decreased virus entry into cells and potentially influenced spike protein expression. Assessment of the stability and recovery potential of N-linked glycan-deficient strains underscored the critical importance of N-glycans at various stages of the virus lifecycle. In vivo experiments infecting piglets with N-glycan-deficient strains exhibited milder clinical symptoms, reduced virus excretion, and less severe pathological lesions compared to conventional strains. These findings offer promising translational applications, proposing N-glycosylation inhibitors as potential therapeutic interventions against PEDV. The utilization of these inhibitors might mitigate virus invasion and disease transmission, providing avenues for effective antiviral strategies and vaccine development. Nonetheless, further research is warranted to elucidate the precise mechanisms of N-linked glycans in PEDV infection for comprehensive clinical applications.

Distinct early role of PTEN regulation during HCMV infection of monocytes.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

High-Affinity-Mediated Viral Entry Triggers Innate Affinity Escape Resulting in Type I IFN Resistance and Impaired T Cell Immunity.

Increased receptor binding affinity may allow viruses to escape from Ab-mediated inhibition. However, how high-affinity receptor binding affects innate immune escape and T cell function is poorly understood. In this study, we used the lymphocytic choriomeningitis virus (LCMV) murine infection model system to create a mutated LCMV exhibiting higher affinity for the entry receptor α-dystroglycan (LCMV-GPH155Y). We show that high-affinity receptor binding results in increased viral entry, which is associated with type I IFN (IFN-I) resistance, whereas initial innate immune activation was not impaired during high-affinity virus infection in mice. Consequently, IFN-I resistance led to defective antiviral T cell immunity, reduced type II IFN, and prolonged viral replication in this murine model system. Taken together, we show that high-affinity receptor binding of viruses can trigger innate affinity escape including resistance to IFN-I resulting in prolonged viral replication.

ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells.

The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection.

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.

Phospho-eIF4E stimulation regulates coronavirus entry by selective expression of cell membrane-residential factors.

The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3, CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3, CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3, CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3, CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3, CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.

IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

Neutralizing antibodies to block viral entry and for identification of entry inhibitors.

Neutralizing antibodies (NAbs) are naturally produced by our immune system to combat viral infections. Clinically, neutralizing antibodies with potent efficacy and high specificity have been extensively used to prevent and treat a wide variety of viral infections, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Immunodeficiency Virus (HIV), Dengue Virus (DENV) and Hepatitis B Virus (HBV). An overwhelmingly large subset of clinically effective NAbs operates by targeting viral envelope proteins to inhibit viral entry into the host cell. Binding of viral envelope protein to the host receptor is a critical rate limiting step triggering a cascade of downstream events, including endocytosis, membrane fusion and pore formation to allow viral entry. In recent years, improved structural knowledge on these processes have allowed researchers to also leverage NAbs as an indispensable tool in guiding discovery of novel antiviral entry inhibitors, providing drug candidates with high efficacy and pan-genus specificity. This review will summarize the latest progresses on the applications of NAbs as effective entry inhibitors and as important tools to develop antiviral therapeutics by high-throughput drug screenings, rational design of peptidic entry inhibitor mimicking NAbs and in silico computational modeling approaches.

Design, synthesis and biological evaluation of novel 1,2,4a,5-tetrahydro-4H-benzo[b][1,4]oxazino[4,3-d][1,4]oxazine-based AAK1 inhibitors with anti-viral property against SARS-CoV-2.

Coronavirus entry into host cells hinges on the interaction between the spike glycoprotein of the virus and the cell-surface receptor angiotensin-converting enzyme 2 (ACE2), initiating the subsequent clathrin-mediated endocytosis (CME) pathway. AP-2-associated protein kinase 1 (AAK1) holds a pivotal role in this pathway, regulating CME by modulating the phosphorylation of the µ subunit of adaptor protein 2 (AP2M1). Herein, we report a series of novel AAK1 inhibitors based on previously reported 1,2,4a,5-tetrahydro-4H-benzo[b] [1,4]oxazino[4,3-d] [1,4]oxazine scaffold. Among 23 synthesized compounds, compound 12e is the most potent one with an IC50 value of 9.38 ± 0.34 nM against AAK1. The in vitro antiviral activity of 12e against SARS-CoV-2 was evaluated using a model involving SARS-CoV-2 pseudovirus infecting hACE2-HEK293 host cells. The results revealed that 12e was superior in vitro antiviral activity against SARS-CoV-2 entry into host cells when compared to SGC-AAK1-1 and LX9211, and its activity was comparable to that of a related and reference compound 8. Mechanistically, all AAK1 inhibitors attenuated AAK1-induced phosphorylation of AP2M1 threonine 156 and disrupted the direct interaction between AP2M1 and ACE2, ultimately inhibiting SARS-CoV-2 infection. Notably, compounds 8 and 12e exhibited a more potent effect in suppressing the phosphorylation of AP2M1 T156 and the interaction between AP2M1 and ACE2. In conclusion, novel AAK1 inhibitor 12e demonstrates significant efficacy in suppressing SARS-CoV-2 infection, and holds promise as a potential candidate for developing novel antiviral drugs against SARS-CoV-2 and other coronavirus infections.

Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells.

INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.