scholarly journals Molecular basis of Coxsackievirus A10 entry using the two-in-one attachment and uncoating receptor KRM1

2020 ◽  
Vol 117 (31) ◽  
pp. 18711-18718
Author(s):  
Yingzi Cui ◽  
Ruchao Peng ◽  
Hao Song ◽  
Zhou Tong ◽  
Xiao Qu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Foot And Mouth Disease ◽  
Atomic Structures ◽  
Viral Particles ◽  
Acidic Conditions ◽  
Underlying Mechanisms ◽  
Viral Protein 1 ◽  
Key Residues

KREMEN1 (KRM1) has been identified as a functional receptor for Coxsackievirus A10 (CV-A10), a causative agent of hand-foot-and-mouth disease (HFMD), which poses a great threat to infants globally. However, the underlying mechanisms for the viral entry process are not well understood. Here we determined the atomic structures of different forms of CV-A10 viral particles and its complex with KRM1 in both neutral and acidic conditions. These structures reveal that KRM1 selectively binds to the mature viral particle above the canyon of the viral protein 1 (VP1) subunit and contacts across two adjacent asymmetry units. The key residues for receptor binding are conserved among most KRM1-dependent enteroviruses, suggesting a uniform mechanism for receptor binding. Moreover, the binding of KRM1 induces the release of pocket factor, a process accelerated under acidic conditions. Further biochemical studies confirmed that receptor binding at acidic pH enabled CV-A10 virion uncoating in vitro. Taken together, these findings provide high-resolution snapshots of CV-A10 entry and identify KRM1 as a two-in-one receptor for enterovirus infection.

5 PRODUCTION OF TRANSGENIC LIVESTOCK USING A LENTIVIRUS EXPRESSING MULTIPLE SHORT INTERFERING RNAs TARGETING FOOT AND MOUTH DISEASE VIRUS

2011 ◽  
Vol 23 (1) ◽  
pp. 109
Author(s):  
M. Peoples ◽  
M. Westhusin ◽  
K. Tessanne ◽  
C. Long
Keyword(s):  
Disease Virus ◽  
Lentiviral Vector ◽  
Tissue Sample ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Antibiotic Selection ◽  
Viral Particles ◽  
Transgenic Livestock ◽  
Mouth Disease Virus

One goal of transgenic livestock production is developing animals with enhanced production characteristics. Transgenic animals with resistance to viral disease could greatly reduce economic losses. The use of short interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting viral genomes have shown great promise in vitro for both human and animal applications. However, because of the rapid mutation rate, viruses are able to escape single siRNA inhibition. One method to reduce the chances of a functional escape virus is to target its genome with multiple shRNAs simultaneously. The goal of this research project was to produce a recombinant lentiviral vector that expresses three unique shRNAs targeting different regions of the foot and mouth disease virus (FMDV) and use it to produce transgenic livestock. In these initial experiments we used the goat as our model system. Previously, we confirmed that three distinct siRNAs individually could reduce the ability of the FMDV virion to replicate in vitro. Based upon these results we produced a recombinant lentiviral vector that utilised three bovine Pol III promoters (7sk, H1, U6-2), each transcribing a different effective shRNA targeting FMDV. In addition, the vector also contained the fluorescent marker zsGreen and an antibiotic resistance gene. The lentiviral vector was co-transfected with pCMV and pMDG into 293T cells to produce replication incompetent retroviral particles. The supernatant was collected and ultra-centrifuged (50 000 × g for 1.5 h) to concentrate the viral particles resulting in a high-titer viral preparation (>109 mL–1 infective viral particles). To produce the transgenic caprine offspring, three embryo donors were superovulated and naturally bred. Nineteen zygotes were surgically collected from the oviduct 24 h after mating. Recombinant lentivirus was microinjected into the zygote perivitelline space. Immediately following the injections, four goat embryos were surgically transferred into the oviduct of each synchronized recipient. Pregnancy was determined by ultrasound at Day 30 in 2 of 5 recipients that received embryos. One pregnancy was carried to term resulting in triplets; 1 live birth, and 2 stillborn. The placenta and tissue sample of the live goat both contained a subpopulation of zsGreen positive cells when analysed with fluorescent microscopy. A fibroblast cell line was derived from the tissue sample and placed under antibiotic selection. Results indicate that only the fluorescent cells also expressed a resistance to antibiotic selection. RNA was collected from the fibroblast cells and mature shRNA production was confirmed using the QuantiMir kit (System Biosystems). Expression of all 3 mature shRNAs was verified in these cells. This data further supports that the entire transgene was integrated into the genome. This is the first report of transgenic livestock produced that expresses multiple shRNAs targeting a viral genome.

scholarly journals Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

2009 ◽  
Vol 83 (21) ◽  
pp. 11275-11282 ◽  
Author(s):  
Stewart Goodwin ◽  
Tobias J. Tuthill ◽  
Armando Arias ◽  
Richard A. Killington ◽  
David J. Rowlands
Keyword(s):  
Disease Virus ◽  
Receptor Binding ◽  
Virus Assembly ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Precursor Protein ◽  
Capsid Precursor ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACT The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in E scherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin αvβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

scholarly journals A Novel Substance Purified from Perilla Frutescens Britton Inhibits an Early Stage of HIV-1 Replication without Blocking Viral Adsorption

2002 ◽  
Vol 13 (5) ◽  
pp. 283-288 ◽  
Author(s):  
T Kawahata ◽  
T Otake ◽  
H Mori ◽  
Y Kojima ◽  
I Oishi ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Entry ◽  
Cytopathic Effect ◽  
Early Stage ◽  
Perilla Frutescens ◽  
Viral Strain ◽  
Viral Particles ◽  
Viral Adsorption ◽  
Hiv 1

Pf-gp6, a 6 kDa anti-degranulation glycoprotein purified from the extract of Perilla frutescens, was examined for its antiviral activity against HIV-1 and HIV-2 in vitro. HIV-1-induced cytopathic effect and proviral DNA synthesis were inhibited in the presence of Pf-gp6. The 50% inhibitory concentrations of Pf-gp6 for various HIV-1 strains, including clinical isolates and CCR5-using (R5) HIV-1, ranged between 1.3 and 71.0 μg/ml, depending on the combination of viral strain and host cell. Furthermore, Pf-gp6 did not directly inactivate infectious viral particles. A time-of-addition experiment revealed that Pf-gp6 lost its activity before zidovudine but after the CXCR-4 antagonist AMD3100 during the early stage of viral infection. Although the pinpoint target of Pf-gp6 remains to be elucidated, it may interfere with a step between viral entry and reverse transcription.

scholarly journals Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry

2008 ◽  
Vol 83 (1) ◽  
pp. 228-240 ◽  
Author(s):  
Barbara Berarducci ◽  
Jaya Rajamani ◽  
Mike Reichelt ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Viral Entry ◽  
Rich Region ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Infected Cells ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-ΔCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.

scholarly journals Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71)

2021 ◽  
Vol 22 (16) ◽  
pp. 8757
Author(s):  
Salima Lalani ◽  
Malihe Masomian ◽  
Chit Laa Poh
Keyword(s):  
Foot And Mouth Disease ◽  
Antiviral Agent ◽  
Underlying Mechanism ◽  
Neurological Complications ◽  
In Vivo Evaluation ◽  
Competitive Binding Assay ◽  
Enterovirus A71 ◽  
Viral Protein 1

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

scholarly journals Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Qi Zhang ◽  
Catherine Zhengzheng Chen ◽  
Manju Swaroop ◽  
Miao Xu ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Drug Repurposing ◽  
Cell Entry ◽  
Entry Pathway ◽  
Viral Particles ◽  
Approved Drugs ◽  
Attachment Factor

Abstract The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

scholarly journals Mechanisms of B-Cell Oncogenesis Induced by Epstein-Barr Virus

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Abhik Saha ◽  
Erle S. Robertson
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Genetically Engineered ◽  
World Population ◽  
Barr Virus ◽  
Viral Particles ◽  
Epstein Barr ◽  
Underlying Mechanisms

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus which asymptomatically infects the majority of the world population. Under immunocompromised conditions, EBV can trigger human cancers of epithelial and lymphoid origin. The oncogenic potential of EBV is demonstrated byin vitroinfection and transformation of quiescent B cells into lymphoblastoid cell lines (LCLs). These cell lines, along with primary infection using genetically engineered viral particles coupled with recent technological advancements, have elucidated the underlying mechanisms of EBV-induced B-cell lymphomagenesis.

scholarly journals Evolutionary Transition toward Defective RNAs That Are Infectious by Complementation

2004 ◽  
Vol 78 (21) ◽  
pp. 11678-11685 ◽  
Author(s):  
Juan García-Arriaza ◽  
Susanna C. Manrubia ◽  
Miguel Toja ◽  
Esteban Domingo ◽  
Cristina Escarmís
Keyword(s):  
Rna Virus ◽  
Foot And Mouth Disease ◽  
Experimental System ◽  
Evolutionary Transition ◽  
Protein Coding ◽  
Coding Regions ◽  
Defective Rnas ◽  
Viral Particles ◽  
Mouth Disease Virus

ABSTRACT Passage of foot-and-mouth disease virus (FMDV) in cell culture resulted in the generation of defective RNAs that were infectious by complementation. Deletions (of nucleotides 417, 999, and 1017) mapped in the L proteinase and capsid protein-coding regions. Cell killing followed two-hit kinetics, defective genomes were encapsidated into separate viral particles, and individual viral plaques contained defective genomes with no detectable standard FMDV RNA. Infection in the absence of standard FMDV RNA was achieved by cotransfection of susceptible cells with transcripts produced in vitro from plasmids encoding the defective genomes. These results document the first step of an evolutionary transition toward genome segmentation of an unsegmented RNA virus and provide an experimental system to compare rates of RNA progeny production and resistance to enhanced mutagenesis of a segmented genome versus its unsegmented counterpart.

scholarly journals LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro

2020 ◽  
Author(s):  
Annika Roth ◽  
Steffen Lütke ◽  
Denise Meinberger ◽  
Gabriele Hermes ◽  
Gerhard Sengle ◽  
...  
Keyword(s):  
Vitamin D ◽  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Viral Entry ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Cell Infection ◽  
S Protein

AbstractObjectiveSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D’s beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects.MethodsRecombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37’s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2.ResultsWe could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated.ConclusionsWe have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

scholarly journals The natural stilbenoid (–)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

2021 ◽  
Author(s):  
Ian Tietjen ◽  
Joel Cassel ◽  
Emery T. Register ◽  
Xiang Yang Zhou ◽  
Troy E. Messick ◽  
...  
Keyword(s):  
South Africa ◽  
United Kingdom ◽  
Receptor Binding ◽  
Viral Entry ◽  
Cytopathic Effect ◽  
Selective Inhibitors ◽  
The United Kingdom ◽  
Pure Compounds ◽  
The Usa

AbstractAntivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host ACE2 receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (–)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (–)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 μM in contrast to an IC50 of 28.3 μM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index = 257.3). When assessed against the USA-WA1/2020 variant, (–)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect assays (IC50 = 10.2 μM) without cytotoxicity. Notably, (–)- hopeaphenol also inhibited two emerging variants of concern originating from the United Kingdom (B.1.1.7) and South Africa (B.1.351) in both cytopathic effect and spike-containing pseudovirus assays with similar (B.1.1.7) or improved (B.1.351) efficacies over the USA- WA1/2020 variant. These results identify (–)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants including those with increased infectivity and/or reduced susceptibility to existing vaccines.ImportanceSARS-CoV-2 antivirals are needed to supplement existing vaccine efforts and target emerging viral variants with increased infectivity or reduced susceptibility to existing vaccines. Here we show that (–)-hopeaphenol, a naturally-occurring stilbenoid compound, in addition to its analogues vatalbinoside A and vaticanol B, inhibit SARS-CoV-2 by blocking the interaction of the viral spike protein with the cellular ACE2 entry receptor. Importantly, in addition to inhibiting the early USA-WA1/2020 SARS-CoV-2 variant, hopeaphenol also inhibits emerging variants of concern including B.1.1.7 (“United Kingdom variant”) and B.1.351 (“South Africa variant”), with improved efficacy against B.1.351. (–)-Hopeaphenol therefore represents a new antiviral lead against infection from multiple SARS-CoV-2 variants.

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