Identification of Key Residues in Subgroup A Avian Leukosis Virus Envelope Determining Receptor Binding Affinity and Infectivity of Cells Expressing Chicken or Quail Tva Receptor

Holmen, Sheri L.; Melder, Deborah C.; Federspiel, Mark J.

doi:10.1128/jvi.75.2.726-737.2001

Cited by 39 publications

(74 citation statements)

References 70 publications

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“…The linear range for a standard assay was between 0.5 and 50 ng of ImmunoPure mouse IgG Fc fragment per ml. Ϫr(log ϫ Ϫ log Kd ) where y is the mean fluorescence; M is the maximum fluorescence; r is the rate; x is the concentration of sTVA-mIgG; and K d is the dissociation constant, defined as the concentration of sTVA-mIgG at half-maximal binding (22).…”

Section: Methodsmentioning

confidence: 99%

“…As hypothesized, several ASLV(A) mutants that had mutations in an envelope glycoprotein variable domain that significantly lowered the binding affinity between the mutant glycoprotein and the quail sTVA-mIgG competitive inhibitor and that escaped receptor interference were selected (22). The viruses contained the E149K, Y142N, or Y142N/E149K mutation in the hr1 variable region in the SU(A) glycoprotein subunit (Fig.…”

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confidence: 99%

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Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

Melder

Pike

VanBrocklin

et al. 2015

J Virol

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The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed. IMPORTANCE A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery.Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes. For enveloped viruses to infect cells, they must fuse their viral membrane with a cellular membrane. For retroviruses, the interaction of the viral envelope glycoproteins with a cellular surface protein receptor initiates the entry and fusion process (1, 2). Retroviral envelope glycopro...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

Melder

Pike

VanBrocklin

et al. 2015

J Virol

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“…Viral spread was monitored by assaying culture supernatants for ALV capsid protein (CA) by ELISA. Virus stocks were generated from cell culture supernatants, and virus titers determined by mixing 10-fold serial dilutions of the virus stock with DF-1 cells and assaying for APpositive infectious units (ifus) 48 h later as described previously (23). DF-1 cells expressing membrane-bound scFvs (∼1 × 10 6 ) and biotinylated laminin were used to estimate laminin-binding affinities by using a FACS-based assay.…”

Section: Methodsmentioning

confidence: 99%

“…The fluorescence from the bound complexes was quantified by using FACS and the results corrected for nonspecific binding of the biotinylated laminin and NeutrAvidin-PE by subtracting the level of fluorescence obtained from the anti-CEA scFv-expressing cells. The maximum mean fluorescence was estimated for each data set by fitting the data via a nonlinear least-squares method to a log logistic growth curve function described previously (23). Only the mutations in the putative hotspots in V H CDR1 and V H CDR2 of L36 scFv reduced the binding affinity for biotinylated laminin by at least 1,000 fold, whereas the mutations in immunoglobulin light chain variable region (VL) V L CDR1 and V L CDR3 had no effect (Fig.…”

Section: Mapping the L36scfv Complementarity Determining Region (Cdr)mentioning

confidence: 99%

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Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

Changming¹,

Pike²,

Rinkoski³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.antibody engineering | avian leukosis virus | protein display technology | antibody binding affinity | avian leukosis virus polypeptide display

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A Single Glycosylation Site Within the Receptor-Binding Domain of the Avian Sarcoma/Leukosis Virus Glycoprotein Is Critical for Receptor Binding

2002

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Retroviral envelope proteins are heavily glycosylated. In some cases, glycosylation has been shown to be important for folding, protein stability, immune evasion, or receptor usage. The receptor-binding subunit (SU or gp85) of the envelope protein (EnvA) of the avian sarcoma/leukosis virus, subtype A (ASLV-A), contains 11 potential N-linked glycosylation sites (NXS/T). To address the importance of N-linked glycosylation for the function of EnvA, we prepared a series of EnvA proteins lacking one or more of these carbohydrate addition sites. Using site-directed mutagenesis, we mutated the S or T in each NXS/T glycosylation sequon to A. We also prepared EnvAs bearing selected double and triple mutations. We examined each mutant EnvA for its ability to be expressed at the cell surface, proteolytically processed into gp85 and gp37, incorporated into MLV pseudotyped virions, and to support infection of cells expressing the ASLV-A receptor, Tva. Eight single mutations were well tolerated, and, in general, EnvA was able to tolerate double mutations of these glycosylation sites. Triple mutations were more variable in their effects. Of the three glycosylation sites important for EnvA function, two are important for folding (EnvA production and processing were severely impaired). For the third, although EnvA processing was impaired, significant amounts of processed EnvA were expressed at the cell surface and incorporated into virions. Nonetheless, this mutant EnvA, EnvADeltaNg10, was unable to support infection. Further examination of EnvADeltaNg10 revealed that it was unable to bind Tva and was severely impaired for binding to a monoclonal antibody which inhibits receptor binding. This work has therefore identified a single N-linked glycosylation site in the SU domain of EnvA that is critical for binding between EnvA and its receptor, Tva.

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Identification of Key Residues in Subgroup A Avian Leukosis Virus Envelope Determining Receptor Binding Affinity and Infectivity of Cells Expressing Chicken or Quail Tva Receptor

Cited by 39 publications

References 70 publications

Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

A Single Glycosylation Site Within the Receptor-Binding Domain of the Avian Sarcoma/Leukosis Virus Glycoprotein Is Critical for Receptor Binding

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