scholarly journals The Avian Coronavirus Infectious Bronchitis Virus Undergoes Direct Low-pH-Dependent Fusion Activation during Entry into Host Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3180-3188 ◽  
Author(s):  
Victor C. Chu ◽  
Lisa J. McElroy ◽  
Vicky Chu ◽  
Beverley E. Bauman ◽  
Gary R. Whittaker
Keyword(s):  
Cell Fusion ◽  
Conformational Changes ◽  
Infectious Bronchitis Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Surface Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Infectious Bronchitis ◽  
Ph Dependent

ABSTRACT Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

scholarly journals The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

2021 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Mariana Marin ◽  
You Zhang ◽  
Fredric S. Cohen ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Fusion ◽  
New World ◽  
Virus Entry ◽  
Low Ph ◽  
Host Cells ◽  
Fusion Pore ◽  
Lassa Virus ◽  
Anionic Lipid ◽  
Late Endosomes ◽  
Ph Dependent

AbstractArenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH in cells expressing human LAMP1 compared to cells lacking this cognate receptor, but this receptor is not absolutely required for virus entry. GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins – a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion is specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. We show that BMP promotes late steps of LASV fusion downstream of hemifusion – the formation and enlargement of fusion pores. This lipid also specifically promotes cell fusion mediated by GPC of the unrelated New World Junin arenavirus. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to direct virus entry to late endosomes.Author SummaryPathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa virus fusion was augmented by the LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

scholarly journals Correlation of membrane protein conformational and functional dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Joel José Montalvo‐Acosta ◽  
George R. Heath ◽  
Yining Jiang ◽  
Xiaolong Gao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
High Temporal Resolution ◽  
Dependent Manner ◽  
Functional Dynamics ◽  
Ph Dependent ◽  
Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

scholarly journals The amino acid at position 624 in the glycoprotein of SFTSV (severe fever with thrombocytopenia virus) plays a critical role in low-pH-dependent cell fusion activity

2017 ◽  
Vol 38 (2) ◽  
pp. 89-97 ◽  
Author(s):  
Yoshimi TSUDA ◽  
Manabu IGARASHI ◽  
Ryo ITO ◽  
Sanae NISHIO ◽  
Kenta SHIMIZU ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Critical Role ◽  
Low Ph ◽  
Dependent Cell ◽  
Ph Dependent ◽  
Severe Fever

scholarly journals The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

2003 ◽  
Vol 77 (5) ◽  
pp. 3058-3066 ◽  
Author(s):  
Laurie J. Earp ◽  
Sue E. Delos ◽  
Robert C. Netter ◽  
Paul Bates ◽  
Judith M. White
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Low Ph ◽  
Target Cells ◽  
Dependent Manner ◽  
Neutral Ph ◽  
Subtype A ◽  
Avian Sarcoma ◽  
Virus Subtype ◽  
Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

scholarly journals The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

2004 ◽  
Vol 78 (1) ◽  
pp. 124-135 ◽  
Author(s):  
A. G. P. Oomens ◽  
Gail W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Low Ph ◽  
Human Respiratory Syncytial Virus ◽  
Dependent Manner ◽  
Practical Applications ◽  
Ph Dependent ◽  
Syncytial Virus

ABSTRACT Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP64/F, a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP64/F, along with two marker ORFs encoding green fluorescent protein (GFP) and β-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSΔsh,g,f/GP64 or RSΔsh,g,f/GP64/F, respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSΔsh [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSΔsh,g,f/GP64 and RSΔsh,g,f/GP64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

scholarly journals Infectious bronchitis virus attaches to lipid rafts and enters cells via clathrin mediated endocytosis

2018 ◽  
Author(s):  
Huan Wang ◽  
Yingjie Sun ◽  
Xiang Mao ◽  
Chunchun Meng ◽  
Lei Tan ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Infectious Bronchitis Virus ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Low Ph ◽  
Infection Process ◽  
Late Endosome ◽  
Poultry Industry ◽  
Infectious Bronchitis ◽  
Late Endosomes

ABSTRACTDue to its economic importance to in poultry industry, the biology and pathogenesis of infectious bronchitis virus (IBV) have been investigated extensively. However, the molecular mechanisms involved in IBV entry are not well characterized. In this study, systematic approaches were used to dissect IBV entry process in various susceptible cells. First, we observed that lipid rafts were involved in IBV attachment. Second, low pH in intracyplasmic vesicles was required for virus entry. By using the specific clathrin mediated endocytosis (CME) inhibitor or knock down of clathrin heavy chain (CHC), we demonstrated that IBV mainly utilized the CME for its entry. Furthermore, GTPase dynamin1 was involved in virus containing vesicle scission and internalization. Surprisingly, CME adaptor Eps15 had no effect on IBV internalization. Third, the penetration of IBV into cells led to active cytoskeleton rearrangement. After internalization, virus particles moved along with the classical endosome/lysosome track, as evidenced by co-localization of R18 labeled IBV with vehicle markers Rab5/Rab7/LAMP1 along with the infection time course. Functional inactivation of Rab5 and Rab7 significantly inhibited IBV infection. VCP, a protein helps early endosome maturation, was involved virus trafficking. Finally, by using the dual R18/DiOC labeled IBV, we observed that membrane fusion with late endosome/lysosome membranes was induced between 2-3 h.p.i.. Taken together, our findings demonstrate that IBV virions attach to lipid rafts and are internalized into cells via CME, move along with early/late endosomes-lysosomes, finally fuse with late endosome-lysosome membranes, release virus genome into cytoplasm. This study provides comprehensive images of IBV attachment-internalization-trafficking-fusion steps.IMPORTANCEIBV, the avian coronavirus isolated in 1937, infects chicken and causes economic loss in poultry industry. It has been reported that the entry of IBV requires low pH. However, the molecular mechanisms underlying IBV internalization and trafficking remain to be clarified. Therefore, we employed multiple chemical and molecular approaches to dissect the entry mechanisms of IBV in susceptible cells. Our results showed IBV entry was significantly inhibited when clathrin-mediated endocytosis (CME) was blocked by chemical inhibitor or depletion of clathrin protein. Moreover, by using R18-labeled IBV, we found that IBV particles attached to lipid rafts, led to actin rearrangement, and moved along with the entire endosomal system. R18/DiOC labeling method showed that IBV fused with late endosomes or lysosomes. This is the first report to describe the entire entry process of IBV, allowing for a better understanding of the infection process of group III avian coronavirus.

scholarly journals N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity

2020 ◽  
Vol 101 (6) ◽  
pp. 599-608
Author(s):  
K. M. Bouwman ◽  
N. Habraeken ◽  
A. Laconi ◽  
A. J. Berends ◽  
L. Groenewoud ◽  
...  
Keyword(s):  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Glycosylation Site ◽  
Host Cells ◽  
Receptor Specificity ◽  
Knock Out ◽  
Recombinant Viruses ◽  
Infectious Bronchitis

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

scholarly journals SARS-CoV-2 infection induces EMT-like molecular changes, including ZEB1-mediated repression of the viral receptor ACE2, in lung cancer models

2020 ◽  
Author(s):  
C. Allison Stewart ◽  
Carl M. Gay ◽  
Kavya Ramkumar ◽  
Kasey R. Cargill ◽  
Robert J. Cardnell ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Epithelial To Mesenchymal Transition ◽  
Surface Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Viral Receptor ◽  
Mesenchymal Transition ◽  
Transcriptional Changes ◽  
Infected Cells ◽  
Potential Strategy

AbstractCOVID-19 is an infectious disease caused by SARS-CoV-2, which enters host cells via the cell surface proteins ACE2 and TMPRSS2. Using normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2. We find that ACE2 expression is restricted to a select population of highly epithelial cells and is repressed by ZEB1, in concert with ZEB1’s established role in promoting epithelial to mesenchymal transition (EMT). Notably, infection of lung cancer cells with SARS-CoV-2 induces metabolic and transcriptional changes consistent with EMT, including upregulation of ZEB1 and AXL, thereby downregulating ACE2 post-infection. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state and lose ACE2 expression, along with its acute respiratory distress syndrome-protective effect, in a ZEB1-dependent manner. AXL-inhibition and ZEB1-reduction, as with bemcentinib, offers a potential strategy to reverse this effect.

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