HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap

Abstract

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Macrophages and HIV-1: dangerous liaisons

“…In HIV-1, the central DNA flap is a 99-nucleotide overlap. This structure enhances the nuclear import of the PIC, increasing the transduction efficiency of lentiviral vectors (Sirven et al, 2000;Zennou et al, 2000). The increased efficiency of cPPT-containing vectors has been attributed at least in part to enhanced nuclear import (Van Maele et al, 2003;Zennou et al, 2000).…”

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

“…The backbone of the lentiviral construct, pTRIP, has been previously described [20]. The vector, pTRIP ΔU3.CMVeGFP [17], expresses the eGFP gene under the control of an internal cytomegalovirus (CMV) promoter.…”

“…recombinant vector as previously described [20]. The supernatants were treated with DNAse I (Roche Diagnostic) prior to ultracentrifugation and the resulting pellet was resuspended in PBS, aliquotted and frozen at −80°C until use.…”

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors

“…[7][8][9][10][11][12][13][14][15][16][17] B-cell precursor (BCP) blasts are very fragile and difficult to maintain alive in culture conditions. 18 The best available results so far for gene transduction have been obtained with second-generation lentiviral vectors monitoring the expression of the CD80 transgene up to 48 h. 5 More recently, second-generation lentiviral vectors have been further modified by deleting a portion of the U3 LTR region in the so-called self-inactivating vector (SIN) with improved safety, 8,9,15,19,20 and by the introduction of two cis-acting elements, one derived from the pol sequence, called central polypurine tract sequence (cPPT), 11,21,22 and the other, termed Wpre, obtained from the genome of the woodchuck hepatitis virus, both believed to increase transgene expression. [23][24][25] As previously demonstrated by our group 26 and others, [27][28][29][30][31][32] CD40 triggering by the CD40L molecule is capable of inducing a number of morphological and functional changes of BCP-ALL blasts, including the upregulation of the surface markers CD40, CD86, CD80, MHC class I and II, CD54 and CD58, and the secretion of chemoattractants MDC and TARC.…”

“…All vectors carry the GFP marker gene and were kindly provided by Dr Naldini, Torino, Italy, and are described elsewhere. [10][11][12][13][14][15][16][17][18][19][20][21][22][23] Viral vector particles were generated by cotransfection of the VSV containing plasmid pMD-G, the packaging construct pCMVdR8.74 and the indicated transfer constructs into 293T cells, followed by determination of concentration of particles by ultracentrifugation, as previously described. 65 Viral titers were evaluated by infecting a known number of REH cells with different volumes of viral vector preparations in a 200 ml volume.…”