Abstract
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.
Highlights
Unintegrated circular extrachromosomal forms of human immunodeficiency virus type 1 (HIV-1)1 DNA are constantly produced during infection of cell culture and in vivo [1,2,3]
After purification by agarose gel electrophoresis, the c1LTR, c2LTR. The final product (c2LTR), supercoiled pLW/C, and pLW/C linearized with PvuI were separately transfected into HeLa cells, either alone or along with an expression plasmid carrying the tat gene of HIV-1
The recovery of p24 in the supernatant was significantly higher in the presence of Tat, which transcriptionally transactivates the HIV-1 long terminal repeat (LTR) [11], indicating that Tat is functional with the extrachromosomal forms of HIV-1 reconstructed in vitro
Summary
Unintegrated circular extrachromosomal forms of human immunodeficiency virus type 1 (HIV-1) DNA are constantly produced during infection of cell culture and in vivo [1,2,3]. The circular forms contain either a single copy or a tandem double copy of the long terminal repeat (LTR) region, which contains the viral transcriptional initiation and termination control elements [4, 5] Despite their nuclear localization [6] and their presence at the site of HIV-1 replication in cell culture [1, 2] and in vivo [3], it is not clear whether the circular forms are able to sustain viral replication. In an attempt to answer this question, we measured the amount of viral p24 protein produced from synthetic circles of HIV-1 DNA constructed to closely mimic the extrachromosomal forms of HIV-1 We show that these forms support the production of viral proteins with varying efficiencies, depending in part upon the relative position of the LTR, and can express infectious virus
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