HIV-1 protein expression from synthetic circles of DNA mimicking the extrachromosomal forms of viral DNA.
We have constructed circular forms of human immunodeficiency virus type 1 viral DNA in vitro that closely resemble the single and double long terminal repeat circular forms of unintegrated viral DNA formed in the nuclei of infected cells. We have analyzed viral protein expression after transient transfection of these circular DNAs into HeLa cells and compared it with expression from a transfected linearized plasmid containing an integrated provirus. Both circular forms are expressed, as judged by the appearance of extracellular p24, and expression is trans-activated by human immunodeficiency virus type 1 Tat. Viral p24 production, however, is approximately an order of magnitude lower than that obtained with transfected integrated viral DNA. Similar data were obtained when a luciferase reporter gene was substituted for the coding regions of the viral DNA. Positional effects of the transcriptional initiation and termination signals in the long terminal repeat appear to account for some of the low expression levels. These data suggest that unintegrated circular viral DNAs are transcriptionally active, although at low levels, and may contribute to overall viral replication in infected people under some conditions.
Cara
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Laboratory of Tumor Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Cereseto
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A
Lori
F
F
Reitz
M S
MS
D001483:Base Sequence; D017931:DNA Primers; D004270:DNA, Circular / Q000138:chemical synthesis / Q000378:metabolism*; D004279:DNA, Viral / Q000138:chemical synthesis / Q000378:metabolism*; D015870:Gene Expression; D016679:Genome, Viral; D016655:HIV Core Protein p24 / Q000096:biosynthesis*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D016662:Virus Integration
D017931:DNA Primers; D004270:DNA, Circular; D004279:DNA, Viral; D016655:HIV Core Protein p24