title: Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.
abstract: The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.
authors.affiliation: Department of Pathology and Immunology, Monash Medical School, Monash University, Melbourne, Victoria, Australia.
authors.forename: C
authors.initials: C
authors.lastname: Bird
authors.forename: J
authors.initials: J
authors.lastname: Burke
authors.forename: P A
authors.initials: PA
authors.lastname: Gleeson
authors.forename: J
authors.initials: J
authors.lastname: McCluskey
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins