Analysis of retroviral assembly using a vaccinia/T7-polymerase complementation system.
The nature of protein-protein interactions during retroviral assembly is not well understood, and mutational analyses of the potential signals involved in the viral assembly process has been difficult, particularly with the avian retroviruses due to the level of viral proteins expressed in the clonal cell lines containing defective viral genomes. We describe here a complementation system in which the retroviral gag/pol and env gene products were expressed independently from different plasmids under the control of the bacteriophage T7 promoter, in avian cells. Coexpression of the T7 polymerase from a vaccinia virus vector resulted in a high level of biosynthesis of retroviral structural proteins and efficient assembly of virus particles. Electron microscopy and protein composition analyses demonstrated that these virions were indistinguishable from those produced from RSV-infected cells. Through the use of mutant glycoprotein genes it was possible to demonstrate the specificity of the assembly process and the applicability of this system to other retroviral systems is described.
Dong
J
J
Department of Microbiology, University of Alabama, Birmingham 35294.
Hunter
E
E
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000254:growth & development; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015686:Gene Products, env / Q000096:biosynthesis; D015683:Gene Products, gag / Q000096:biosynthesis; D015684:Gene Products, pol / Q000096:biosynthesis; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D016093:Mason-Pfizer monkey virus / Q000235:genetics / Q000254:growth & development; D011784:Quail; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D013045:Species Specificity; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins; D014771:Virion / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure
D015686:Gene Products, env; D015683:Gene Products, gag; D015684:Gene Products, pol; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases