scholarly journals Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in cis or in trans by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein

2001 ◽  
Vol 75 (8) ◽  
pp. 3685-3695 ◽  
Author(s):  
Dimitri Lavillette ◽  
Bertrand Boson ◽  
Stephen J. Russell ◽  
François-Loı̈c Cosset
Keyword(s):  
Membrane Fusion ◽  
Receptor Binding ◽  
Leukemia Virus ◽  
Binding Domain ◽  
Surface Glycoprotein ◽  
Receptor Binding Domain ◽  
Domain Interactions ◽  
Carboxy Terminal ◽  
Leukemia Viruses ◽  
Murine Leukemia

ABSTRACT Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in trans by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in cis, via the proper RBD of the viral Env itself, or in trans, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers.

scholarly journals Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

2001 ◽  
Vol 75 (23) ◽  
pp. 11851-11862 ◽  
Author(s):  
S. Michael Rothenberg ◽  
Mari N. Olsen ◽  
Louise Chang Laurent ◽  
Rachel Adams Crowley ◽  
Patrick O. Brown
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Receptor Binding Domain ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Env Protein ◽  
Carboxy Terminal ◽  
Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

scholarly journals Role of Variable Regions A and B in Receptor Binding Domain of Amphotropic Murine Leukemia Virus Envelope Protein

1998 ◽  
Vol 72 (11) ◽  
pp. 9101-9108 ◽  
Author(s):  
Jin-Young Han ◽  
Yi Zhao ◽  
W. French Anderson ◽  
Paula M. Cannon
Keyword(s):  
Receptor Binding ◽  
Envelope Protein ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Variable Regions ◽  
Amino Terminal ◽  
Amphotropic Murine Leukemia Virus ◽  
Murine Leukemia

ABSTRACT For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.

scholarly journals Envelope Proteins Containing Single Amino Acid Substitutions Support a Structural Model of the Receptor-Binding Domain of Bovine Leukemia Virus Surface Protein

2002 ◽  
Vol 76 (21) ◽  
pp. 10861-10872 ◽  
Author(s):  
Elizabeth R. Johnston ◽  
Lorraine M. Albritton ◽  
Kathryn Radke
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Bovine Leukemia Virus ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Receptor Binding Domain ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of env transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.

Role of N-linked Glycosylation in the Activity of the Friend Murine Leukemia Virus SU Protein Receptor-Binding Domain

Virology ◽  
1994 ◽  
Vol 202 (1) ◽  
pp. 496-499 ◽  
Author(s):  
Jean-Luc Battini ◽  
Samuel C. Kayman ◽  
Abraham Pinter ◽  
Jean Michel Heard ◽  
Olivier Danos
Keyword(s):  
Receptor Binding ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Protein Receptor ◽  
Murine Leukemia

scholarly journals An Aromatic Side Chain Is Required at Residue 8 of SU for Fusion of Ecotropic Murine Leukemia Virus

2004 ◽  
Vol 78 (1) ◽  
pp. 508-512 ◽  
Author(s):  
Zhaohui Qian ◽  
Lorraine M. Albritton
Keyword(s):  
Membrane Fusion ◽  
Aromatic Ring ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Glycoprotein ◽  
Side Chain ◽  
Complete Fusion ◽  
Aromatic Side Chain ◽  
Fusion Pores ◽  
Murine Leukemia

ABSTRACT The surface glycoprotein (SU) of most gammaretroviruses contains a conserved histidine at its amino terminus. In ecotropic murine leukemia virus SU, replacement of histidine 8 with arginine (H8R) or deletion of H8 (H8del) abolishes infection and cell-cell fusion but has no effect on binding to the cellular receptor. We report here that an aromatic ring side chain is essential to the function of residue 8. The size of the aromatic ring appears to be important, as does its ability to form a hydrogen bond. In addition, infection by all of the nonaromatic amino acid substitutions could be partially rescued by the addition of two suppressor mutations (glutamine 227 to arginine [Q227R] and aspartate 243 to tyrosine [D243Y]) or by exposure to chlorpromazine, an agent that induces fusion pores in hemifusion intermediates to complete fusion, suggesting that, like the previously described H8R mutant, the mutants reported here also arrest membrane fusion at the hemifusion state. We propose that H8 is a key switch-point residue in the conformation changes that lead to membrane fusion and present a possible mechanism for how its substitution arrests fusion at the hemifusion state.

scholarly journals Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses

2000 ◽  
Vol 74 (2) ◽  
pp. 899-913 ◽  
Author(s):  
Lucille O'Reilly ◽  
Monica J. Roth
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Cell Types ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Rich Region ◽  
C Terminus ◽  
Nih 3T3 ◽  
Proline Rich Region ◽  
Murine Leukemia

ABSTRACT Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

scholarly journals In-silico design of a multi-epitope recombinant vaccine against SARS-CoV-2 targeting the receptor binding domain

2020 ◽  
Author(s):  
Harshawardhan Pande
Keyword(s):  
T Cell ◽  
B Cell ◽  
Receptor Binding ◽  
In Silico ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Recombinant Vaccine ◽  
Binding Domain ◽  
Surface Glycoprotein ◽  
Receptor Binding Domain

The COVID-19 pandemic caused by the SARS-CoV-2 virus is posing a major global challenge due to its rapid infectivity and lethality. Despite a global effort towards creating a vaccine, no viable vaccine currently exists. While multiple bioinformatic studies have attempted to predict epitopes, they have focused on the whole spike protein without considering antibody mediated enhancement or Th-2 immunopathology and have missed some important but less antigenic epitopes in the receptor binding domain. Therefore, this study used in silico methods to design and evaluate a potential multiepitope vaccine that specifically targets the receptor binding domain due to its critical function in viral entry. Immunoinformatic tools were used to specifically examine the receptor binding domain of the surface glycoprotein for suitable T cell and B cell epitopes. The selected 5 B cell and 8 T cell epitopes were then constructed into a subunit vaccine and appropriate adjuvants along with the universal immunogenic PADRE sequence were added to boost efficacy. The structure of the vaccine construct was predicted through a de novo approach and molecular docking simulations were performed which demonstrated high affinity binding to TLR 5 receptor and appropriate HLA proteins. Finally, the vaccine candidate was cloned into an expression vector for use as a recombinant vaccine. Similarities to some recent epitope mapping studies suggest a high potential for eliciting neutralizing antibodies and generating a favorable overall immune response.

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