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While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV. However, hepatocytes expressing NTCP exhibit varying susceptibilities to HBV infection. This study aimed to investigate whether other host factors modulate the process of HBV infection. Liver biopsy samples obtained from children with hepatitis B were used for single-cell sequencing and susceptibility analysis. Primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice were used to analyze the effect of candidate host factors on HBV infection. Single-cell sequencing and susceptibility analysis revealed a positive correlation between neuropilin-1 (NRP1) expression and HBV infection. In the HBV-infected cell model, NRP1 overexpression before HBV inoculation significantly enhanced viral attachment and internalization, and promoted viral infection in the presence of NTCP. Mechanistic studies indicated that NRP1 formed a complex with LHBs (large hepatitis B surface proteins) and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of LHBs. This NRP1-preS1 interaction subsequently promoted the binding of preS1 to NTCP, facilitating viral infection. Moreover, disruption of the NRP1-preS1 interaction by the NRP1 antagonist EG00229 significantly attenuated the binding affinity between NTCP and preS1, thereby inhibiting HBV infection both in vitro and in vivo. Our findings indicate that NRP1 is a novel host factor for HBV infection, which interacts with preS1 and NTCP to modulate HBV entry into hepatocytes. HBV infection is a global public health problem, but the understanding of the early infection process of HBV remains limited. Through single-cell sequencing, we identified a novel host factor, NRP1, which modulates HBV entry by interacting with HBV preS1 and NTCP. Moreover, antagonists targeting NRP1 can inhibit HBV infection both in vitro and in vivo. This study could further advance our comprehension of the early infection process of HBV. [Display omitted] • Single-cell sequencing and susceptibility analysis identify host factors involved in the process of HBV infection. • NRP1 enhances viral attachment and internalization, and promotes viral infection in the presence of NTCP. • NRP1 modulates HBV infection by interacting with HBV preS1 and NTCP. • NRP1 antagonist EG00229 treatment pre-inoculation inhibits HBV infection in cell model and human liver-chimeric mice. [ABSTRACT FROM AUTHOR]

Human coronavirus (CoV) HKU1 infection typically causes common cold but can lead to pneumonia in children, older people, and immunosuppressed individuals. Recently, human transmembrane serine protease 2 (hTMPRSS2) was identified as the functional receptor for HKU1, but its region and residues critical for HKU1 S binding remain elusive. In this study, we find that HKU1 could utilize human and hamster, but not rat, mouse, or bat TMPRSS2 for virus entry, displaying a narrow host range. Using human-bat TMPRSS2 chimeras, we show that the serine peptidase (SP) domain of TMPRSS2 is essential for entry of HKU1. Further extensive mutagenesis analyses of the C-terminal regions of SP domains of human and bat TMPRSS2s identify residues 417 and 469 critical for entry of HKU1. Replacement of either D417 or Y469 with asparagine in hTMPRSS2 abolishes its abilities to mediate entry of HKU1 S pseudovirions and cell-cell fusion, whereas substitution of N417 with D or N469 with Y in bat TMPRSS2 (bTMPRSS2) renders it supporting HKU1 entry. Our findings contribute to a deeper understanding of coronavirus-receptor interactions and cross-species transmission. [ABSTRACT FROM AUTHOR]

Rabies virus causes an estimated 59,000 annual fatalities worldwide and promising therapeutic treatments are necessary to develop. In this study, affinity tag-purification mass spectrometry was employed to delineate RABV glycoprotein and host protein interactions, and PDIA3/ERP57 was identified as a potential inhibitor of RABV infection. PDIA3 restricted RABV infection with follow mechanisms: PDIA3 mediated the degradation of RABV G protein by targeting lysine 332 via the selective macroautophagy/autophagy pathway; The PDIA3 interactor, AP3B1 (adaptor related protein complex 3 subunit beta 1) was indispensable in PDIA3-triggered selective degradation of the G protein; Furthermore, PDIA3 competitively bound with NCAM1/NCAM (neural cell adhesion molecule 1) to block RABV G, hindering viral entry into host cells. PDIA3 190-199 aa residues bound to the RABV G protein were necessary and sufficient to defend against RABV. These results demonstrated the therapeutic potential of biologics that target PDIA3 or utilize PDIA3 190-199 aa peptide to treat clinical rabies. Abbreviation: aa: amino acids; ANXA2: annexin A2; AP-MS: affinity tag purification-mass spectrometry; AP3B1: adaptor related protein complex 3 subunit beta 1; ATP6V1A: ATPase H+ transporting V1 subunit A; ATP6V1H: ATPase H+ transporting V1 subunit H; BafA1: bafilomycin A1; CHX: cycloheximide; co-IP: co-immunoprecipitation; DDX17: DEAD-box helicase 17; DmERp60: drosophila melanogaster endoplasmic reticulum p60; EBOV: Zaire ebolavirus virus; EV: empty vector; GANAB: glucosidase II alpha subunit; G protein: glycoprotein; GRM2/mGluR2: glutamate metabotropic receptor 2; HsPDIA3: homo sapiens protein disulfide isomerase family A member 3; IAV: influenza virus; ILF2: interleukin enhancer binding factor 2; KO: knockout; MAGT1: magnesium transporter 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MmPDIA3: mus musculus protein disulfide isomerase associated 3; NCAM1/NCAM: neural cell adhesion molecule 1; NGFR/p75NTR: nerve growth factor receptor; NGLY1: N-glycanase 1; OTUD4: OTU deubiquitinase 4; PDI: protein disulfide isomerase; PPIs: protein-protein interactions; RABV: rabies virus; RUVBL2: RuvB like AAA ATPase 2; SCAMP3: secretory carrier membrane protein 3; ScPdi1: Saccharomyces cerevisiae s288c protein disulfide isomerase 1; SLC25A6: solute carrier family 25 member 6; SQSTM1/p62: sequestosome 1; VSV: vesicular stomatitis virus. [ABSTRACT FROM AUTHOR]

BACKGROUND: Viral infection elicits the type I interferon (IFN-I) response in host cells and subsequently inhibits viral infection through inducing hundreds of IFN-stimulated genes (ISGs) that counteract many steps in the virus life cycle. However, most of ISGs have unclear functions and mechanisms in viral infection. Thus, more work is required to elucidate the role and mechanisms of individual ISGs against different types of viruses. RESULTS: Herein, we demonstrate that poliovirus receptor-like protein4 (PVRL4) is an ISG strongly induced by IFN-I stimulation and various viral infections. Overexpression of PVRL4 protein broadly restricts growth of enveloped RNA and DNA viruses, including vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whereas deletion of PVRL4 in host cells increases viral infections. Mechanistically, it suppresses viral entry by blocking viral-cellular membrane fusion through inhibiting endosomal acidification. The vivo studies demonstrate that Pvrl4-deficient mice were more susceptible to the infection of VSV and IAV. CONCLUSION: Overall, our studies not only identify PVRL4 as an intrinsic broad-spectrum antiviral ISG, but also provide a candidate host-directed target for antiviral therapy against various viruses including SARS-CoV-2 and its variants in the future.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron entry involves spike (S) glycoprotein-mediated fusion of viral and late endosomal membranes. Here, using single-molecule Förster resonance energy transfer (sm-FRET) imaging and biochemical measurements, we directly visualized conformational changes of individual spike trimers on the surface of SARS-CoV-2 Omicron pseudovirions during fusion activation. We observed that the S2 domain of the Omicron spike is a dynamic fusion machine. S2 reversibly interchanges between the pre-fusion conformation and two previously undescribed intermediate conformations. Acidic pH shifts the conformational equilibrium of S2 toward an intermediate conformation and promotes the membrane hemi-fusion reaction. Moreover, we captured conformational reversibility in the S2 domain, which suggests that spike can protect itself from pre-triggering. Furthermore, we determined that Ca2+ directly promotes the S2 conformational change from an intermediate conformation to post-fusion conformation. In the presence of a target membrane, low pH and Ca2+ stimulate the irreversible transition to S2 post-fusion state and promote membrane fusion. [Display omitted] • Omicron spike fusion domain S2 shows dynamic plasticity • S2 spontaneously samples two functional intermediate conformations to fusion pathway • Low pH promotes formation of fusion competent intermediate conformations • Ca2+ catalyzes an S2 conformational change to a stable post fusion conformation at low pH Dey et al. used single molecule FRET for a real-time visualization of SARS-CoV-2 spike conformational dynamics on the virion surface during fusion triggered by low pH and Ca2+. The intermediates identified during dynamic S protein mediated fusion provide mechanistic insights for Omicron entry that may guide CoV inhibitor design. [ABSTRACT FROM AUTHOR]

The Ebola virus is a deadly pathogen that has been threatening public health for decades. Recent studies have revealed alternative viral invasion routes where Ebola virus approaches cells via interactions among phosphatidylserine (PS), PS binding ligands such as Gas6, and TAM family receptors such as Axl. In this study, we investigate the interactions among phosphatidylserine on the Ebola viral-like particle (VLP) membrane, human Gas6, and human Axl using atomic force microscope-based single molecule force spectroscopy to compare their binding strength and affinity from a biomechanical perspective. The impact of calcium ions on their interactions is also studied and quantified to provide more details on the calcium-dependent phosphatidylserine-Gas6 binding mechanism. Our results indicate that, in the presence of calcium ions, the binding strengths of VLP-Gas6 and VLP-Gas6-Axl increase but are still weaker than that of Gas6-Axl, and the binding affinity of VLP-Gas6 and VLP-Gas6-Axl is largely improved. The binding strength and affinity of Gas6-Axl basically remain the same, indicating no impact in the presence of calcium ions. Together, our study suggests that, under physiological conditions with calcium present, the Ebola virus can utilize its membrane phosphatidylserine to dock on cell surface via Gas6-Axl bound complex. [ABSTRACT FROM AUTHOR]

Human coronavirus 229E (HCoV-229E) is an endemic coronavirus responsible for approximately one-third of "common cold" cases. To infect target cells, HCoV-229E first binds to its receptor on the cell surface and then can follow different pathways, entering by direct fusion or by taking advantage of host cell mechanisms such as endocytosis. Based on the role of clathrin, the process can be classified into clathrin-dependent or -independent endocytosis. This study characterizes the role of clathrin-mediated endocytosis (CME) in HCoV-229E infection of the human hepatoma cell line Huh-7. Using specific CME inhibitory drugs, we demonstrated that blocking CME significantly reduces HCoV-229E infection. Additionally, CRISPR/Cas9-mediated knockout of the µ subunit of adaptor protein complex 2 (AP-2) further corroborated the role of CME, as KOs showed over a 50% reduction in viral infection. AP-2 plays an important role in clathrin recruitment and the maturation of clathrin-coated vesicles. Our study also confirmed that in Huh-7 cells, HCoV-229E requires endosomal acidification for successful entry, as viral entry decreased when treated with lysomotropic agents. Furthermore, the colocalization of HCoV-229E with early endosome antigen 1 (EEA-1), only present in early endosomes, suggested that the virus uses an endosomal route for entry. These findings highlight, for the first time, the role of CME in HCoV-229E infection and confirm previous data of the use of the endosomal route at a low pH in the experimental cell model Huh-7. Our results provide new insights into the mechanisms of entry of HCoV-229E and provide a new basis for the development of targeted antiviral therapies. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry‐associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV‐2), Middle East respiratory syndrome coronavirus (MERS‐CoV), human coronavirus 229E (HCoV‐229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin‐converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta‐galactoside alpha‐2,6‐sialyltransferase 1 (ST6GAL1), and beta‐galactoside alpha‐2,3‐sialyltransferase 4 (ST3GAL4) were selected as key entry‐associated genes for SARS‐CoV‐2, HCoV‐229E, MERS‐CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air–liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real‐time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry‐associated genes showed distinct regulation patterns in response to each interleukin‐4 (IL‐4), interleukin‐13 (IL‐13), tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL‐4 and IL‐13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF‐α and IFN‐γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN‐γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS‐CoV‐2 and HCoV‐229E infections but higher susceptibility to MERS‐CoV and influenza infections. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive efforts, the mechanism underlying virus entry for PEDV remains elusive. In this study, we first identified PEDV-susceptible and non-susceptible cell lines by using PEDV spike pseudotyped vesicular stomatitis virus. Subsequently, we conducted a comprehensive transcriptomic analysis on these cell lines. Through integrating differential expression gene analysis with weighted gene co-expression network analysis, we identified the key pathways that are correlated with the PEDV entry. Our analysis revealed a strong correlation between cholesterol, sterols, and lipid transport with PEDV entry, suggesting a potential role for cholesterol transport in the PEDV entry. For further investigation, we treated Huh7, Vero and LLC-PK1 cells with a cholesterol transport inhibitor, ezetimibe, and observed a significant inhibition of PEDV entry and subsequent viral replication in these cells. Interestingly, pre-treating Huh7 cells with ezetimibe resulted in an increase in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses. Moreover, we found that cholesterol could facilitate the entry of PEDV into Huh7 and Vero cells, and this promoting effect can be blocked by ezetimibe. These findings suggest that targeting cholesterol transport specifically inhibits PEDV entry into susceptible cells. Our study offers novel insights into the mechanism of PEDV entry and the development of new therapeutic strategies against this economically important virus.

Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited. This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

Abstract The orf virus (ORFV) poses a serious threat to the health of domestic small ruminants (i.e., sheep and goats) and humans on a global scale, causing around $150 million in annual losses to livestock industry. However, the host factors involved in ORFV infection and replication are still elusive. In this study, we compared the RNA-seq profiles of ORFV-infected or non-infected sheep testicular interstitial cells (STICs) and identified a novel host gene, potassium voltage-gated channel subfamily E member 4 (KCNE4), as a key host factor involved in the ORFV infection. Both RNA-seq data and RT-qPCR assay revealed a significant increase in the expression of KCNE4 in the infected STICs from 9 to 48 h post infection (hpi). On the other hand, the RT-qPCR assay detected a decrease in ORFV copy number in both the STICs transfected by KCNE4 siRNA and the KCNE4 knockout (KO) HeLa cells after the ORFV infection, together with a reduced fluorescence ratio of ORFV-GFP in the KO HeLa cells at 24 hpi, indicating KCNE4 to be critical for the ORFV infection. Furthermore, the attachment and internalization assays showed decreased ORFV attachment, internalization, replication, and release by the KO HeLa cells, demonstrating a potential inhibition of ORFV entry into the cells by KCNE4. Pretreatment with the KCNE4 inhibitors such as quinidine and fluoxetine significantly repressed the ORFV infection. All our findings reveal KCNE4 as a novel host regulator of the ORFV entry and replication, shedding new insight into the interactive mechanism of ORFV infection. The study also highlights the K+ channels as possible druggable targets to impede viral infection and disease.

The orf virus (ORFV) poses a serious threat to the health of domestic small ruminants (i.e., sheep and goats) and humans on a global scale, causing around $150 million in annual losses to livestock industry. However, the host factors involved in ORFV infection and replication are still elusive. In this study, we compared the RNA-seq profiles of ORFV-infected or non-infected sheep testicular interstitial cells (STICs) and identified a novel host gene, potassium voltage-gated channel subfamily E member 4 (KCNE4), as a key host factor involved in the ORFV infection. Both RNA-seq data and RT-qPCR assay revealed a significant increase in the expression of KCNE4 in the infected STICs from 9 to 48 h post infection (hpi). On the other hand, the RT-qPCR assay detected a decrease in ORFV copy number in both the STICs transfected by KCNE4 siRNA and the KCNE4 knockout (KO) HeLa cells after the ORFV infection, together with a reduced fluorescence ratio of ORFV-GFP in the KO HeLa cells at 24 hpi, indicating KCNE4 to be critical for the ORFV infection. Furthermore, the attachment and internalization assays showed decreased ORFV attachment, internalization, replication, and release by the KO HeLa cells, demonstrating a potential inhibition of ORFV entry into the cells by KCNE4. Pretreatment with the KCNE4 inhibitors such as quinidine and fluoxetine significantly repressed the ORFV infection. All our findings reveal KCNE4 as a novel host regulator of the ORFV entry and replication, shedding new insight into the interactive mechanism of ORFV infection. The study also highlights the K+ channels as possible druggable targets to impede viral infection and disease. [ABSTRACT FROM AUTHOR]

One of the entry mechanisms of the SARS-CoV-2 coronavirus into host cells involves endosomal acidification. It has been proposed that under acidic conditions, the fusion peptide proximal region (FPPR) of the SARS-CoV-2 spike glycoprotein acts as a pH-dependent switch, modulating immune response accessibility by influencing the positioning of the receptor binding domain (RBD). This would provide indirect coupling of RBD opening to the environmental pH. Here, we explored this possible pH-dependent conformational equilibrium of the FPPR within the SARS-CoV-2 spike glycoprotein. We analyzed hundreds of experimentally determined spike structures from the Protein Data Bank and carried out pH-replica exchange molecular dynamics to explore the extent to which the FPPR conformation depends on pH and the positioning of the RBD. A meta-analysis of experimental structures identified alternate conformations of the FPPR among structures in which this flexible regions was resolved. However, the results did not support a correlation between the FPPR conformation and either RBD position or the reported pH of the cryo-EM experiment. We calculated pKa values for titratable side chains in the FPPR region using PDB structures, but these pKa values showed large differences between alternate PDB structures that otherwise adopt the same FPPR conformation type. This hampers the comparison of pKa values in different FPPR conformations to rationalize a pH-dependent conformational change. We supplemented these PDB-based analyses with all-atom simulations and used constant-pH replica exchange molecular dynamics to estimate pKa values in the context of flexibility and explicit water. The resulting titration curves show good reproducibility between simulations, but they also suggest that the titration curves of the different FPPR conformations are the same within the error bars. In summary, we were unable to find evidence supporting the previously published hypothesis of an FPPR pH-dependent equilibrium: neither from existing experimental data nor from constant-pH MD simulations. The study underscores the complexity of the spike system and opens avenues for further exploration into the interplay between pH and SARS-CoV-2 viral entry mechanisms. [ABSTRACT FROM AUTHOR]

Background: Alphaviruses are a diverse group of pathogens that have garnered considerable attention due to their impact on human health. By investigating alphavirus receptors, researchers can elucidate viral entry mechanisms and gain important clues for the prevention and treatment of viral diseases. This study presents an in-depth analysis of the research progress made in the field of alphavirus receptors through bibliometric analysis. Methods: This study encompasses various aspects, including historical development, annual publication trends, author and cited-author analysis, institutional affiliations, global distribution of research contributions, reference analysis with strongest citation bursts, keyword analysis, and a detailed exploration of recent discoveries in alphavirus receptor research. Results: The results of this bibliometric analysis highlight key milestones in alphavirus receptor research, demonstrating the progression of knowledge in this field over time. Additionally, the analysis reveals current research hotspots and identifies emerging frontiers, which can guide future investigations and inspire novel therapeutic strategies. Conclusion: This study provides an overview of the state of the art in alphavirus receptor research, consolidating the existing knowledge and paving the way for further advancements. By shedding light on the significant developments and emerging areas of interest, this study serves as a valuable resource for researchers, clinicians, and policymakers engaged in combating alphavirus infections and improving public health. [ABSTRACT FROM AUTHOR]

COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2. In this new screen, rutaecarpine (an alkaloid from Evodia rutaecarpa) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration. [ABSTRACT FROM AUTHOR]

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.IMPORTANCEEnveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

ABSTRACT: Newcastle disease virus, a member of the Paramyxoviridae family, causes significant economic losses in poultry worldwide. To identify novel antiviral agents against NDV, 36 canthin-6-one analogs were evaluated in this study. Our data showed that 8 compounds exhibited excellent inhibitory effects on NDV replication with IC50 values in the range of 5.26 to 11.76 μM. Besides, these analogs inhibited multiple NDV strains with IC50 values within 12 μM and exerted antiviral activity against peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). Among these analogs, 16 presented the strongest anti-NDV activity (IC50 = 5.26 μM) and minimum cytotoxicity (CC50 > 200 μM) in DF-1 cells. Furthermore, 16 displayed antiviral activity in different cell lines. Our results showed that 16 did not affect the viral adsorption while it can inhibit the entry of NDV by suppressing the Akt pathway. Further study found that 16-treatment inhibited the NDV-activated ERK pathway, thereby promoting the expression of interferon-related genes. Our findings reveal an antiviral mechanism of canthin-6-one analogs through inhibition of the Akt and ERK signaling pathways. These results point to the potential value of canthin-6-one analogs to serve as candidate antiviral agents for NDV.

ABSTRACT Infection with respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in young children and older people. Despite intensive efforts over the past few decades, no direct-acting small-molecule agents against RSV are available. Most small-molecule candidates targeting the RSV fusion (F) protein pose a considerable risk of inducing drug-resistant mutations. Here, we explored the in vitro and in vivo virological properties of the K394R variant, a cross-resistant mutant capable of evading multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in vitro and more pathogenic than the parental strain in vivo. The small molecule (2E,2′E)-N,N′-((1R,2S,3S)−3-hydroxycyclohexane-1,2-diyl)bis(3-(2-bromo-4-fluorophenyl) acrylamide) (CL-A3-7), a structurally optimized compound derived from a natural caffeoylquinic acid derivative, substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant. Mechanistically, CL-A3-7 significantly inhibited virus–cell fusion during RSV entry by blocking the interaction between the viral F protein and the cellular insulin-like growth factor 1 receptor (IGF1R). Collectively, these results indicate severe disease risks caused by the K394R variant and reveal a new anti-RSV mechanism to overcome K394R-associated resistance.IMPORTANCERespiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance.

Emerging highly pathogenic viruses can pose profound impacts on global health, the economy, and society. To meet that challenge, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) centers for early-stage identification and validation of novel antiviral drug candidates against viruses with pandemic potential. As part of this initiative, we established paired entry assays that simultaneously screen for inhibitors specifically targeting SARS-CoV-2 (SARS2), Lassa virus (LASV) and Machupo virus (MACV) entry. To do so we employed a dual pseudotyped virus (PV) infection system allowing us to screen ∼650,000 compounds efficiently and cost-effectively. Adaptation of these paired assays into 1536 well-plate format for ultra-high throughput screening (uHTS) resulted in the largest screening ever conducted in our facility, with over 2.4 million wells completed. The paired infection system allowed us to detect two PV infections simultaneously: LASV + MACV, MACV + SARS2, and SARS2 + LASV. Each PV contains a different luciferase reporter gene which enabled us to measure the infection of each PV exclusively, albeit in the same well. Each PV was screened at least twice utilizing different reporters, which allowed us to select the inhibitors specific to a particular PV and to exclude those that hit off targets, including cellular components or the reporter proteins. All assays were robust with an average Z’ value ranging from 0.5 to 0.8. The primary screening of ∼650,000 compounds resulted in 1812, 1506, and 2586 unique hits for LASV, MACV, and SARS2, respectively. The confirmation screening narrowed this list further to 60, 40, and 90 compounds that are unique to LASV, MACV, and SARS2, respectively. Of these compounds, 8, 35, and 50 compounds showed IC50 value < 10 μM, some of which have much greater potency and excellent antiviral activity profiles specific to LASV, MACV, and SARS2, and none are cytotoxic. These selected compounds are currently being studied for their mechanism of action and to improve their specificity and potency through chemical modification.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a highly lethal zoonotic virus with a potential large-scale outbreak, which poses a great threat to world health and security. In order to explore more potential factors associated with NiV, a proximity labeling method was applied to investigate the F, G, and host protein interactions systematically. We screened 1996 and 1524 high-confidence host proteins that interacted with the NiV fusion (F) glycoprotein and attachment (G) glycoprotein in HEK293T cells by proximity labeling technology, and 863 of them interacted with both F and G. The results of GO and KEGG enrichment analysis showed that most of these host proteins were involved in cellular processes, molecular binding, endocytosis, tight junction, and other functions. Cytoscape software (v3.9.1) was used for visual analysis, and the results showed that Cortactin (CTTN), Serpine mRNA binding protein 1 (SERBP1), and stathmin 1 (STMN1) were the top 20 proteins and interacted with F and G, and were selected for further validation. We observed colocalization of F-CTTN, F-SERBP1, F-STMN1, G-CTTN, G-SERBP1, and G-STMN1 using confocal fluorescence microscopy, and the results showed that CTTN, SERBP1, and STMN1 overlapped with NiV F and NiV G in HEK293T cells. Further studies found that CTTN can significantly inhibit the infection of the Nipah pseudovirus (NiVpv) into host cells, while SERBP1 and STMN1 had no significant effect on pseudovirus infection. In addition, CTTN can also inhibit the infection of the Hendra pseudovirus (HeVpv) in 293T cells. In summary, this study revealed that the potential host proteins interacted with NiV F and G and demonstrated that CTTN could inhibit NiVpv and HeVpv infection, providing new evidence and targets for the study of drugs against these diseases. [ABSTRACT FROM AUTHOR]

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1's involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) binds sialic acid receptors on the cell surface to enter the host cells, which is the key step in initiating infection, transmission and pathogenesis. Understanding the factors that contribute to the highly efficient entry of IAV into human cells will help elucidate the mechanism of viral entry and pathogenicity, and provide new targets for intervention. In the present study, we reported a novel membrane protein, C1QTNF5, which binds to the hemagglutinin protein of IAV and promotes IAV infection in vitro and in vivo. We found that the HA1 region of IAV hemagglutinin is critical for the interaction with C1QTNF5 protein, and C1QTNF5 interacts with hemagglutinin mainly through its N-terminus (1-103 aa). In addition, we further demonstrated that overexpression of C1QTNF5 promotes IAV entry, while blocking the interaction between C1QTNF5 and IAV hemagglutinin greatly inhibits viral entry. However, C1QTNF5 does not function as a receptor to mediate IAV infection in sialic acid-deficient CHO-Lec2 cells, but promotes IAV to attach to these cells, suggesting that C1QTNF5 is an important attachment factor for IAV. This work reveals C1QTNF5 as a novel IAV attachment factor and provides a new perspective for antiviral strategies. [ABSTRACT FROM AUTHOR]

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackieadenovirus 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. [ABSTRACT FROM AUTHOR]

ABSTRACT Numerous coreceptors have been shown to facilitate hACE2-dependent or hACE2-independent infection by SARS-CoV-2. A recent study published in mBio by Yu et al. showed that the histamine receptor H1 (HRH1) functions as an alternative receptor for SARS-CoV-2 via direct binding to viral spike proteins (F. Yu, X. Liu, H. Ou, X. Li, et al., mBio e01088-24, 2024, https://doi.org/10.1128/mbio.01088-24). Furthermore, they present compelling evidence that antihistamine drugs targeting HRH1 potently inhibit SARS-CoV-2 entry. This study highlights the therapeutic potential of repurposable antihistamines against COVID-19.

ABSTRACT Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating broad viral tropism and diversified druggable potential. We and others have found that antihistamine drugs, particularly histamine receptor H1 (HRH1) antagonists, potently inhibit SARS-CoV-2 infection. In this study, we provided compelling evidence that HRH1 acts as an alternative receptor for SARS-CoV-2 by directly binding to the viral spike protein. HRH1 also synergistically enhanced hACE2-dependent viral entry by interacting with hACE2. Antihistamine drugs effectively prevent viral infection by competitively binding to HRH1, thereby disrupting the interaction between the spike protein and its receptor. Multiple inhibition assays revealed that antihistamine drugs broadly inhibited the infection of various SARS-CoV-2 mutants with an average IC50 of 2.4 µM. The prophylactic function of these drugs was further confirmed by authentic SARS-CoV-2 infection assays and humanized mouse challenge experiments, demonstrating the therapeutic potential of antihistamine drugs for combating coronavirus disease 19.IMPORTANCEIn addition to human angiotensin-converting enzyme 2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can utilize alternative cofactors to facilitate viral entry. In this study, we discovered that histamine receptor H1 (HRH1) not only functions as an independent receptor for SARS-CoV-2 but also synergistically enhances ACE2-dependent viral entry by directly interacting with ACE2. Further studies have demonstrated that HRH1 facilitates the entry of SARS-CoV-2 by directly binding to the N-terminal domain of the spike protein. Conversely, antihistamine drugs, primarily HRH1 antagonists, can competitively bind to HRH1 and thereby prevent viral entry. These findings revealed that the administration of repurposable antihistamine drugs could be a therapeutic intervention to combat coronavirus disease 19.

Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus's life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an urgent need for effective therapeutic options. SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic that has resulted in significant morbidity and mortality worldwide. The virus is known to enter host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, and emerging evidence suggests that heparan sulfate proteoglycans (HSPGs) play a crucial role in facilitating this process. HSPGs are abundant cell surface proteoglycan present in many tissues, including the lung, and have been shown to interact directly with the spike protein of SARS-CoV-2. This review aims to summarize the current understanding of the role of HSPGs in SARS-CoV-2 infection and the potential of developing new therapies targeting HSPGs.

ABSTRACT Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities toward the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step.IMPORTANCEMany efforts in small-molecule screens have been made to counter SARS-CoV-2 infection by targeting the viral main protease, the major element that processes viral proteins after translation. Here, we discovered that calpain inhibitors further block SARS-CoV-2 infection in a main protease-independent manner. We identified the host cysteine protease calpain-2 as an important positive regulator of the cell surface levels of SARS-CoV-2 cellular receptor ACE2 and, thus, a facilitator of viral infection. By either pharmacological inhibition or genetic knockout of calpain-2, the SARS-CoV-2 binding to host cells is blocked and viral infection is decreased. Our findings highlight a novel mechanism of ACE2 regulation, which presents a potential new therapeutic target. Since calpain inhibitors also potently interfere with the viral main protease, our data also provide a mechanistic understanding of the potential use of calpain inhibitors as dual inhibitors (entry and replication) in the clinical setting of COVID-19 diseases. Our findings bring mechanistic insights into the cellular process of SARS-CoV-2 entry and offer a novel explanation to the mechanism of activities of calpain inhibitors.

Pseudorabies virus (PRV), a herpesvirus responsible for Aujeszky's disease, causes high mortality in swine populations. To develop effective and novel antiviral strategies, it is essential to understand the mechanism of entry used by PRV to infect its host. Viruses have different ways of entering host cells. Among others, they can use endocytosis, a fundamental cellular process by which substances from the external environment are internalized into the cell. This process is classified into clathrin-mediated endocytosis (CME) and clathrinindependent endocytosis (CIE), depending on the role of clathrin. Although the involvement of cholesterol-rich lipid rafts in the entry of PRV has already been described, the importance of other endocytic pathways involving clathrin remains unexplored to date. Here, we characterize the role of CME in PRV entry into the PK15 swine cell line. By using CME inhibitory drugs, we report a decrease in PRV infection when the CME pathway is blocked. We also perform the shRNA knockdown of the μ-subunit of the adaptor protein AP-2 (AP2M1), which plays an important role in the maturation of clathrin-coated vesicles, and the infection is greatly reduced when this subunit is knocked down. Furthermore, transmission electron microscopy images report PRV virions inside clathrincoated vesicles. Overall, this study suggests for the first time that CME is a mechanism used by PRV to enter PK15 cells and provides valuable insights into its possible routes of entry. [ABSTRACT FROM AUTHOR]

Umifenovir is a broad‐spectrum antiviral agent used to treat influenza in China and Russia, and it has been studied as an antiviral agent for the treatment of coronavirus disease 2019 (COVID‐19). We have previously reported the synthesis of novel umifenovir analogues and their biological evaluation with a focus on their inhibitory activity against the binding of the spike glycoprotein (S‐protein) of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and the angiotensin‐converting enzyme 2 (ACE2) receptor; however, no strong inhibitory activity was observed from these analogues. In the present study, an additional set of umifenovir analogues was synthesized with replacement of the substituents at the 2‐, 3‐, and 4‐positions of the indole, and a cell‐based assay using SARS‐CoV‐2 (B.1.1) was performed to examine the antiviral activity of the analogues. We found that one of the newly synthesized umifenovir analogues exhibited antiviral activity and reduced the viral load to 0.06 % as compared to the control when it was assessed in the presence of nafamostat and marimastat, which inhibit cell‐surface viral entry. In contrast, when this analogue was evaluated without the addition of nafamostat or marimastat, it exhibited less antiviral activity, suggesting that the umifenovir analogue would exert antiviral activity mainly by inhibiting endosome‐mediated viral entry. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) is the primary causative agent of influenza, colloquially called the flu. Each year, it infects up to a billion people, resulting in hundreds of thousands of human deaths, and causes devastating avian outbreaks with worldwide losses worth billions of dollars. Always present is the possibility that a highly pathogenic novel subtype capable of direct human-to-human transmission will spill over into humans, causing a pandemic as devastating if not more so than the 1918 influenza pandemic. While antiviral drugs for influenza do exist, they target very few aspects of IAV replication and risk becoming obsolete due to antiviral resistance. Antivirals targeting other areas of IAV replication are needed to overcome this resistance and combat the yearly epidemics, which exact a serious toll worldwide. This review aims to summarise the key steps in the IAV replication cycle, along with highlighting areas of research that need more focus. [ABSTRACT FROM AUTHOR]

Henipaviruses are a genus of emerging pathogens that includes the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of disease. Henipaviruses rely on two surface glycoproteins, known as the attachment and fusion proteins, to facilitate entry into host cells. As new and divergent members of the genus have been discovered and structurally characterized, key differences and similarities have been noted. This review surveys the available structural information on Henipavirus glycoproteins, complementing this with information from related biophysical and structural studies of the broader Paramyxoviridae family of which Henipaviruses are members. The process of viral entry is a primary focus for vaccine and drug development, and this review aims to identify critical knowledge gaps in our understanding of the mechanisms that drive Henipavirus fusion. [ABSTRACT FROM AUTHOR]

Avian metapneumovirus subgroup C (aMPV/C) causes respiratory diseases and egg dropping in chickens and turkeys, resulting in severe economic losses to the poultry industry worldwide. Integrin β1 (ITGB1), a transmembrane cell adhesion molecule, is present in various cells and mediates numerous viral infections. Herein, we demonstrate that ITGB1 is essential for aMPV/C infection in cultured DF-1 cells, as evidenced by the inhibition of viral binding by EDTA blockade, Arg-Ser-Asp (RSD) peptide, monoclonal antibody against ITGB1, and ITGB1 short interfering (si) RNA knockdown in cultured DF-1 cells. Simulation of the binding process between the aMPV/C fusion (F) protein and avian-derived ITGB1 using molecular dynamics showed that ITGB1 may be a host factor benefiting aMPV/C attachment or internalization. The transient expression of avian ITGB1-rendered porcine and feline non-permissive cells (DQ cells and CRFK cells, respectively) is susceptible to aMPV/C infection. Kinetic replication of aMPV/C in siRNA-knockdown cells revealed that ITGB1 plays an important role in aMPV/C infection at the early stage (attachment and internalization). aMPV/C was also able to efficiently infect human non-small cell lung cancer (A549) cells. This may be a consequence of the similar structures of both metapneumovirus F protein-specific motifs (RSD for aMPV/C and RGD for human metapneumovirus) recognized by ITGB1. Overexpression of avian-derived ITGB1 and human-derived ITGB1 in A549 cells enhanced aMPV/C infectivity. Taken together, this study demonstrated that ITGB1 acts as an essential receptor for aMPV/C attachment and internalization into host cells, facilitating aMPV/C infection. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease in pigs. The low-density lipoprotein receptor (LDLR) is a transcriptional target of the sterol-regulatory element-binding proteins (SREBPs) and participates in the uptake of LDL-derived cholesterol. However, the involvement of LDLR in PRV infection has not been well characterized. We observed an increased expression level of LDLR mRNA in PRV-infected 3D4/21, PK-15, HeLa, RAW264.7, and L929 cells. The LDLR protein level was also upregulated by PRV infection in PK-15 cells and in murine lung and brain. The treatment of cells with the SREBP inhibitor, fatostatin, or with SREBP2-specific small interfering RNA prevented the PRV-induced upregulation of LDLR expression as well as viral protein expression and progeny virus production. This suggested that PRV activated SREBPs to induce LDLR expression. Furthermore, interference in LDLR expression affected PRV proliferation, while LDLR overexpression promoted it. This indicated that LDLR was involved in PRV infection. The study also demonstrated that LDLR participated in PRV invasions. The overexpression of LDLR or inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR and targets it for lysosomal degradation, significantly enhanced PRV attachment and entry. Mechanistically, LDLR interacted with PRV on the plasma membrane, and pretreatment of cells with LDLR antibodies was able to neutralize viral entry. An in vivo study indicated that the treatment of mice with the PCSK9 inhibitor SBC-115076 promoted PRV proliferation. The data from the study indicate that PRV hijacks LDLR for viral entry through the activation of SREBPs. IMPORTANCE Pseudorabies virus (PRV) is a herpesvirus that primarily manifests as fever, pruritus, and encephalomyelitis in various domestic and wild animals. Owing to its lifelong latent infection characteristics, PRV outbreaks have led to significant financial setbacks in the global pig industry. There is evidence that PRV variant strains can infect humans, thereby crossing the species barrier. Therefore, gaining deeper insights into PRV pathogenesis and developing updated strategies to contain its spread are critical. This study posits that the low-density lipoprotein receptor (LDLR) could be a co-receptor for PRV infection. Hence, strategies targeting LDLR may provide a promising avenue for the development of effective PRV vaccines and therapeutic interventions. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) has been known as a common viral pathogen that can infect several parts of the body, leading to various clinical manifestations. According to this diverse manifestation, HSV-1 infection in many cell types was demonstrated. Besides the HSV-1 cell tropism, e.g., fibroblast, epithelial, mucosal cells, and neurons, HSV-1 infections can occur in human T lymphocyte cells, especially in activated T cells. In addition, several studies found that actin polymerization and filopodia formation support HSV-1 infection in diverse cell types. Hence, the goal of this review is to explore the mechanism of HSV-1 infection in various types of cells involving filopodia formation and highlight potential future directions for HSV-1 entry-related research. Moreover, this review covers several strategies for possible anti-HSV drugs focused on the entry step, offering insights into potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus‐encoded spike protein and fusion between the viral and target cell membranes. Here, we report that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor‐binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity in vitro. These receptor‐like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc‐gamma receptor I, a chimera of their antigen‐binding fragment with the transmembrane domain of ACE2 or a membrane‐bound B cell receptor, indicating that ACE2 and its specific interaction with the spike protein are dispensable for SARS‐CoV‐2 entry. These results suggest that antibody responses against SARS‐CoV‐2 may help expand the viral tropism to otherwise nonpermissive cell types with potential implications for viral transmission and pathogenesis. Synopsis: SARS‐CoV‐2 enters host cells by first engaging its cellular receptor to induce membrane fusion. Certain monoclonal neutralizing antibodies can independently function as a receptor to support viral infectivity in cultured cells.Monoclonal neutralizing antibodies can serve as an entry receptor for SARS‐CoV‐2 in cell culture.Receptor‐like antibodies can function in three different membrane‐bound forms.The cognate receptor ACE2 is dispensable for SARS‐CoV‐2 entry in vitro. [ABSTRACT FROM AUTHOR]

Rotavirus (RV) is one of the most significant pathogens in humans and animals with diarrhea worldwide. Cell entry is the first step in viral infection, and the outer capsid protein VP4 is crucial for RV attachment and internalization. In order to discover novel candidate host factors involved in RV cell entry, a proximity labeling method was applied to systematically investigate the VP4 and host protein interactions. A total of 174 high-confidence host proteins were identified using proximity labeling. Further analysis showed that 88 proteins were located in the cytoskeleton, plasma membrane, and extracellular region, which could be involved in RV entry. Importantly, vimentin (VIM) and actin-related protein 2 (ACTR2) were identified to promote RV infection at an early step. The results of co-immunoprecipitation assay showed that VIM or ACTR2 physically interacted with VP4. Blocking VIM or ACTR2 function by silencing with small interfering RNA or inhibition by specific antibodies significantly restricted RV infection. Furthermore, increasing the amounts of VIM or ACTR2 by overexpression from transfected recombinant proteins or incubation with recombinant proteins promoted RV infection. Collectively, this study revealed that RV VP4 interacted with host proteins and demonstrated that interaction with VIM and ACTR2 promoted RV replication, providing valuable resources and potential drug targets for better understanding and treating this disease. [ABSTRACT FROM AUTHOR]

We recently discovered a superantigen-like motif sequentially and structurally similar to a staphylococcal enterotoxin B (SEB) segment, near the S1/S2 cleavage site of the SARS-CoV-2 spike protein, which might explain the multisystem inflammatory syndrome (MIS-C) observed in children and the cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif at its polybasic (PRRA) insert to inhibit infection in live virus assays. The overlap between the superantigenic site of the spike and its proteolytic cleavage site suggests that the mAb prevents viral entry by interfering with the proteolytic activity of cell proteases (furin and TMPRSS2). The high affinity of 6D3 for this site originates from a polyacidic segment at its heavy chain CDR2. The study points to the potential utility of 6D3 for possibly treating COVID-19, MIS-C, or common colds caused by human coronaviruses that also possess a furin-like cleavage site.

Pseudorabies virus (PRV), a herpesvirus responsible for Aujeszky’s disease, causes high mortality in swine populations. To develop effective and novel antiviral strategies, it is essential to understand the mechanism of entry used by PRV to infect its host. Viruses have different ways of entering host cells. Among others, they can use endocytosis, a fundamental cellular process by which substances from the external environment are internalized into the cell. This process is classified into clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE), depending on the role of clathrin. Although the involvement of cholesterol-rich lipid rafts in the entry of PRV has already been described, the importance of other endocytic pathways involving clathrin remains unexplored to date. Here, we characterize the role of CME in PRV entry into the PK15 swine cell line. By using CME inhibitory drugs, we report a decrease in PRV infection when the CME pathway is blocked. We also perform the shRNA knockdown of the μ-subunit of the adaptor protein AP-2 (AP2M1), which plays an important role in the maturation of clathrin-coated vesicles, and the infection is greatly reduced when this subunit is knocked down. Furthermore, transmission electron microscopy images report PRV virions inside clathrin-coated vesicles. Overall, this study suggests for the first time that CME is a mechanism used by PRV to enter PK15 cells and provides valuable insights into its possible routes of entry.