scholarly journals The roles of two major domains of the porcine deltacoronavirus spike subunit 1 in receptor binding and neutralization

2021 ◽  
Author(s):  
Yan Liu ◽  
Bin Wang ◽  
Qi-Zhang Liang ◽  
Fang-Shu Shi ◽  
Chun-Miao Ji ◽  
...  
Keyword(s):  
Amino Acids ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Aromatic Amino Acids ◽  
Intervention Strategies ◽  
Aminopeptidase N ◽  
Interspecies Transmission ◽  
Spike Glycoprotein ◽  
Glycosylation Sites

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host-cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated that overexpression of APN orthologues from various species including mice and felines in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo , distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1 B ) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both the domain A (S1 A ) and S1 B were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1 A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1 A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. Importance Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro . Here we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1 A and S1 B ) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1 B domain critical for APN binding, and demonstrating neutralization activity of S1 A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV’s great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

scholarly journals Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7629-7640 ◽  
Author(s):  
Sonia Navas-Martin ◽  
Susan T. Hingley ◽  
Susan R. Weiss
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Receptor Binding ◽  
Amino Acid Substitution ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Functional Compensation

ABSTRACT Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L- R654H -H716D- E1035D ) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

Structural characterization, tissue distribution, and functional expression of murine aminoacylase III

2004 ◽  
Vol 286 (4) ◽  
pp. C848-C856 ◽  
Author(s):  
Alexander Pushkin ◽  
Gerardo Carpenito ◽  
Natalia Abuladze ◽  
Debra Newman ◽  
Vladimir Tsuprun ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Mass ◽  
Rotational Symmetry ◽  
Aromatic Amino Acids ◽  
Functional Expression ◽  
Proximal Tubules ◽  
Size Exclusion ◽  
Northern Analysis ◽  
Mercapturic Acids

Many xenobiotics are detoxified through the mercapturate metabolic pathway. The final product of the pathway, mercapturic acids ( N-acetylcysteine S-conjugates), are secreted predominantly by renal proximal tubules. Mercapturic acids may undergo a transformation mediated by aminoacylases and cysteine S-conjugate β-lyases that leads to nephrotoxic reactive thiol formation. The deacetylation of cysteine S-conjugates of N-acyl aromatic amino acids is thought to be mediated by an aminoacylase whose molecular identity has not been determined. In the present study, we cloned aminoacylase III, which likely mediates this process in vivo, and characterized its function and structure. The enzyme consists of 318 amino acids and has a molecular mass (determined by SDS-PAGE) of ∼35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is ∼140 kDa as determined by size-exclusion chromatography, which suggests that it is a tetramer. In agreement with this hypothesis, transmission electron microscopy and image analysis of aminoacylase III showed that the monomers of the enzyme are arranged with a fourfold rotational symmetry. Northern analysis demonstrated an ∼1.4-kb transcript that was expressed predominantly in kidney and showed less expression in liver, heart, small intestine, brain, lung, testis, and stomach. In kidney, aminoacylase III was immunolocalized predominantly to the apical domain of S1 proximal tubules and the cytoplasm of S2 and S3 proximal tubules. The data suggest that in kidney proximal tubules, aminoacylase III plays an important role in deacetylating mercapturic acids. The predominant cytoplasmic localization of aminoacylase III may explain the greater sensitivity of the proximal straight tubule to the nephrotoxicity of mercapturic acids.

scholarly journals Expression of Hemagglutinin Esterase Protein from Recombinant Mouse Hepatitis Virus Enhances Neurovirulence

2005 ◽  
Vol 79 (24) ◽  
pp. 15064-15073 ◽  
Author(s):  
Lubna Kazi ◽  
Arjen Lissenberg ◽  
Richard Watson ◽  
Raoul J. de Groot ◽  
Susan R. Weiss
Keyword(s):  
Receptor Binding ◽  
Demyelinating Disease ◽  
Hepatitis Virus ◽  
Critical Role ◽  
Spike Glycoprotein ◽  
Murine Hepatitis Virus ◽  
Recombinant Viruses ◽  
Viral Spread ◽  
The Central Nervous System

ABSTRACT Murine hepatitis virus (MHV) infection provides a model system for the study of hepatitis, acute encephalitis, and chronic demyelinating disease. The spike glycoprotein, S, which mediates receptor binding and membrane fusion, plays a critical role in MHV pathogenesis. However, viral proteins other than S also contribute to pathogenicity. The JHM strain of MHV is highly neurovirulent and expresses a second spike glycoprotein, the hemagglutinin esterase (HE), which is not produced by MHV-A59, a hepatotropic but only mildly neurovirulent strain. To investigate a possible role for HE in MHV-induced neurovirulence, isogenic recombinant MHV-A59 viruses were generated that produced either (i) the wild-type protein, (ii) an enzymatically inactive HE protein, or (iii) no HE at all (A. Lissenberg, M. M. Vrolijk, A. L. W. van Vliet, M. A. Langereis, J. D. F. de Groot-Mijnes, P. J. M. Rottier, and R. J. de Groot, J. Virol. 79:15054-15063, 2005 [accompanying paper]). A second, mirror set of recombinant viruses was constructed in which, in addition, the MHV-A59 S gene had been replaced with that from MHV-JHM. The expression of HE in combination with A59 S did not affect the tropism, pathogenicity, or spread of the virus in vivo. However, in combination with JHM S, the expression of HE, regardless of whether it retained esterase activity or not, resulted in increased viral spread within the central nervous system and in increased neurovirulence. Our findings suggest that the properties of S receptor utilization and/or fusogenicity mainly determine organ and host cell tropism but that HE enhances the efficiency of infection and promotes viral dissemination, at least in some tissues, presumably by serving as a second receptor-binding protein.

scholarly journals Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

2010 ◽  
Vol 84 (13) ◽  
pp. 6846-6860 ◽  
Author(s):  
Nadi T. Wickramasekera ◽  
Paula Traktman
Keyword(s):  
Amino Acids ◽  
Structure Function ◽  
Protein Interactions ◽  
Function Analysis ◽  
Aromatic Amino Acids ◽  
Protein Protein Interactions ◽  
Core Component ◽  
Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

scholarly journals Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

2021 ◽  
Vol 17 (3) ◽  
pp. e1009356
Author(s):  
Takaaki Koma ◽  
Cheng Huang ◽  
Adrian Coscia ◽  
Steven Hallam ◽  
John T. Manning ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rational Design ◽  
Critical Role ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Wild Type ◽  
Virus Growth ◽  
Glycosylation Sites

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

scholarly journals Enhancement of SARS-CoV-2 Receptor Binding Domain -CR3022 Human Antibody Binding Affinity via in Silico Engineering Approach

2020 ◽  
Author(s):  
Fateme Sefid ◽  
Zahra Payandeh ◽  
Ghasem Azamirad ◽  
Behzad Mansoori ◽  
Behzad Baradaran ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Affinity Maturation ◽  
Binding Domain ◽  
Human Antibody ◽  
Receptor Binding Domain ◽  
Receptor Binding Site ◽  
Specificity And Sensitivity

Abstract Background: The nCoV-2019 is a cause of COVID-19 disease. The surface spike glycoprotein (S), which is necessary for virus entry through the intervention of the host receptor and it mediates virus-host membrane fusion, is the primary coronavirus antigen (Ag). The angiotensin-converting enzyme 2 (ACE2) is reported to be the effective human receptor for SARS-CoVs 2. ACE2 receptor can be prevented by neutralizing antibodies (nAbs) such as CR3022 targeting the virus receptor-binding site. Considering the importance of computational docking, and affinity maturation we aimed to find the important amino acids of the CR3022 antibody (Ab). These amino acids were then replaced by other amino acids to improve Ab-binding affinity to a receptor-binding domain (RBD) of the 2019-nCoV spike protein. Finally, we measured the binding affinity of Ab variants to the Ag. Result: Our findings disclosed that several variant mutations could successfully improve the characteristics of the Ab binding compared to the normal antibodies. Conclusion: The modified antibodies may be possible candidates for stronger affinity binding to Ags which in turn can affect the specificity and sensitivity of antibodies.

scholarly journals Identification of a Receptor-Binding Domain of the Spike Glycoprotein of Human Coronavirus HCoV-229E

2003 ◽  
Vol 77 (4) ◽  
pp. 2530-2538 ◽  
Author(s):  
Aurelio Bonavia ◽  
Bruce D. Zelus ◽  
David E. Wentworth ◽  
Pierre J. Talbot ◽  
Kathryn V. Holmes
Keyword(s):  
Amino Acids ◽  
Receptor Binding ◽  
Protein A ◽  
Binding Domain ◽  
Expression Vectors ◽  
Receptor Binding Domain ◽  
3T3 Cells ◽  
Spike Glycoprotein ◽  
Human Coronavirus ◽  
S Proteins

ABSTRACT Human coronavirus HCoV-229E uses human aminopeptidase N (hAPN) as its receptor (C. L. Yeager et al., Nature 357:420-422, 1992). To identify the receptor-binding domain of the viral spike glycoprotein (S), we expressed soluble truncated histidine-tagged S glycoproteins by using baculovirus expression vectors. Truncated S proteins purified by nickel affinity chromatography were shown to be glycosylated and to react with polyclonal anti-HCoV-229E antibodies and monoclonal antibodies to the viral S protein. A truncated protein (S547) that contains the N-terminal 547 amino acids bound to 3T3 mouse cells that express hAPN but not to mouse 3T3 cells transfected with empty vector. Binding of S547 to hAPN was blocked by an anti-hAPN monoclonal antibody that inhibits binding of virus to hAPN and blocks virus infection of human cells and was also blocked by polyclonal anti-HCoV-229E antibody. S proteins that contain the N-terminal 268 or 417 amino acids did not bind to hAPN-3T3 cells. Antibody to the region from amino acid 417 to the C terminus of S blocked binding of S547 to hAPN-3T3 cells. Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor.

Fluxes and membrane transport of amino acids in rat liver under different protein diets

1990 ◽  
Vol 259 (5) ◽  
pp. E614-E625 ◽  
Author(s):  
P. Fafournoux ◽  
C. Remesy ◽  
C. Demigne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acids ◽  
High Protein Diet ◽  
High Protein ◽  
Protein Diet ◽  
Transport Systems ◽  
Low Concentrations ◽  
Significant Uptake

The aim of the present work was to evaluate in vivo the role of the transport step in hepatic amino acid metabolism. To vary hepatic utilization of amino acids, rats were adapted to diets containing various concentrations of casein (5, 15, and 60%). In rats fed 5 or 15% casein diets, Gln and Glu were released by the liver, and there was a significant uptake of Ala. Hepatic fluxes of amino acids increased considerably after adaptation to high-casein diet (up to 1.55 mumol.min-1.g liver-1 for Ala), because of the rise in afferent concentrations as well as enhanced uptake percentage (peaking at 60–75% for most glucogenic amino acids). Adaptation to a high-protein diet led to induction of not only system A but also of most of the other transport systems (Gly, anionic, T, y+, and to a lesser extent system N); only systems ASC and L were unchanged. The study of amino acid repartition between liver and plasma with different diets indicates that transport could modulate utilization of Ala, Ser, Thr, Gly, Gln, and Asp. For Arg and Asn, present in very low concentrations in liver under any condition, the transport step should be the major locus of control of their metabolism. For amino acids chiefly transported by nonconcentrative systems, such as aromatic amino acids, cellular metabolism could also be limited by the transport process. In conclusion, during adaptation to a high-protein diet, there is apparently a coordinated adaptation of amino acid transport and of their intracellular metabolism. For some amino acids, induction of catabolic enzymes seems greater than that of transport, so that the transport step may play an important role in control of metabolic fluxes. For example, concentration of amino acids such as Thr may be markedly depressed in rats adapted to a high-protein diet.

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