Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72.
A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.
García-Escudero
R
R
Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid), Facultad de Ciencias, Cantoblanco, Spain.
Andrés
G
G
Almazán
F
F
Viñuela
E
E
D000358:African Swine Fever Virus / Q000235:genetics* / Q000648:ultrastructure; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D009024:Morphogenesis; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D014709:Vero Cells; D014771:Virion / Q000378:metabolism
D001426:Bacterial Proteins; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C063745:capsid protein p72, African swine fever virus; D007544:Isopropyl Thiogalactoside