Class I lentiviral integrase mutants reveal cell-cycle dependent expression from persistent unintegrated proviral DNA: implications for HIV-1 persistence in vivo

Dyana Saenz, Nils Loewen, David Leske, Margaret Good, Jonathan Holmes & Eric M. Poeschla - Molecular Medicine Program, Mayo Medical School, 200 First Street SW, Rochester (USA)

Background: Unintegrated HIV-1 DNA forms are prevalent in vivo, but the circumstances under which this DNA is available for transcription and expression are unclear. Recently Tat and Nef expression from un-integrated HIV-1 DNA in resting T cells has been detected, and replication of class I integrase (IN) mutant HIV-1 has been observed in some proliferating cell lines. Material and Methods: We constructed and validated the first class I integrase mutants (D66V and D118A) for a nonprimate lentivirus (FIV), as well as analogous HIV-1 integrase mutants, and studied the cell cycle dependence of viral and viral vector-mediated expression. We documented class I properties, and performed extensive molecular characterization in vivo and in tissue culture with viruses and subgenomic vectors. Results: D66V and D118A mutants of FIV integrase had class I properties, whether analysed alone or combined: Gag/Pol precursor expression, proteolytic processing, particle formation, and RT production were normal.  However, transduction of dividing fibroblasts was blocked (5 log reduction, RT-normalized to wild-type), and southern blotting showed integration was prevented. When RT-normalized stocks were injected into rat retinae, expression was widespread in retinal pigment epithelium at 3 months for wild-type (WT), compared with only rare positive cells for D66V vectors. However, we also observed entirely equivalent transduction of non-dividing, primary post-mitotic rat neurons (retinal ganglion cells) with class I-mutant and WT vectors. Investigating this result further, we found that transgene expression in growth-arrested fibroblasts (aphidicolin, 15 µg/ml) was not inhibited at all by class I mutations (97% vs. 99% of cells transduced with WT vector at MOI = 5, comparing RT-normalized stocks), and expression level per cell was equal to or greater than WT. Similar class I properties and cell cycle-dependent expression were seen with Class I mutant HIV. Cells were then transduced under aphidicolin arrest, and released from the block 48 hours later. Release of cells 99% transduced with WT FIV or HIV vectors at 48 hours resulted in stable transgene expression, durable to repeated passage, in 40-80% of cells. In contrast, release of cells transduced with equal RT units of class I IN mutant FIV or HIV vectors resulted in steady decline of expression, from 97% to 0% of cells by day 10.  Southern blots showed lack of integration irrespective of cell cycle, and increased levels of 2-LTR circles for the class I mutants in growth-arrested cells. Further analyses of LTR-directed expression are in progress. Conclusions: Unintegrated FIV and HIV-1 vector DNAs are capable of high-level expression from internal promoters and this expression is equivalent to that of integrated DNA if cells are growth-arrested. Expression levels correlate with abundant, persistent 2-LTR circles.