Dataset Information

Baculovirus Expression and Potential Diagnostic Application of the Gp51 Envelope Glycoprotein of Genetic Mutants of the Bovine Leukaemia Virus.

ABSTRACT: Introduction:Field isolates of bovine leukaemia virus (BLV) show the presence of a few amino acid substitutions in major conformational G and H epitopes on surface glycoprotein gp51. Potentially, these substitutions can affect the 3D structure of these epitopes leading to their diminished immunoreactivity. The aim of this study was to express three gp51 glycoproteins carrying mutated epitopes as recombinant baculovirus proteins in insect cells to test their immunoreactivity with bovine sera. Material and Methods:Env gene chimeras encoding mutated epitopes G and H in the env backbone of BLV FLK strain were constructed, cloned into pFastBac1 vector, and expressed in baculovirus. Results:The presence of recombinant gp51 protein in Sf9 insect cells was confirmed using monoclonal antibodies. ELISA tests were developed to check the immunoreactivity of recombinant protein with bovine sera. Conclusion:Recombinant gp51 proteins with altered G and H epitopes can be used for further studies to analyse the serological response of bovine sera towards BLV antigenic variants.

SUBMITTER: Rola-Luszczak M

PROVIDER: S-EPMC6458546 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Baculovirus Expression and Potential Diagnostic Application of the Gp51 Envelope Glycoprotein of Genetic Mutants of the Bovine Leukaemia Virus.

Rola-Łuszczak Marzena M Grabowska Agnieszka A Szewczyk Bogusław B Kuźmak Jacek J

Journal of veterinary research 20190322 1

<h4>Introduction</h4>Field isolates of bovine leukaemia virus (BLV) show the presence of a few amino acid substitutions in major conformational G and H epitopes on surface glycoprotein gp51. Potentially, these substitutions can affect the 3D structure of these epitopes leading to their diminished immunoreactivity. The aim of this study was to express three gp51 glycoproteins carrying mutated epitopes as recombinant baculovirus proteins in insect cells to test their immunoreactivity with bovine s ...[more]

PMID: 30989129

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Project description:The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation.IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.

Dataset Information

Baculovirus Expression and Potential Diagnostic Application of the Gp51 Envelope Glycoprotein of Genetic Mutants of the Bovine Leukaemia Virus.

Publications

Baculovirus Expression and Potential Diagnostic Application of the Gp51 Envelope Glycoprotein of Genetic Mutants of the Bovine Leukaemia Virus.

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