HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore


Authors: Nathalie J Arhel, Sylvie Souquere-Besse, Sandie Munier, Philippe Souque, Stephanie Guadagnini, Sandra Tutherford, Marie-Christine Prevost, Terry D Allen, Pierre Charneau 

EMBOJ (2007) 26: 3025-3037

The HIV1 central DNA Flap acts as a cisacting determinant of HIV1 genome nuclear import. Indeed, DNAFlap reinsertion within lentiviralderived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV1 nuclear import. Here, we show that reverse transcription (RT°) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate postfusion event, but rather occurs at the nuclear pore upon RT° completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV1 RT°, acts as a viral promoting element for the uncoating of HIV1 at the nuclear pore.


Lentiviruses have the unique ability among retroviruses to integrate and replicate efficiently in nondividing target cells (Gartner et al, 1986; Weinberg et al, 1991). The active nuclear import of their genome through the nuclear membrane is the key to mitosisindependent lentiviral replication.

We have previously shown (Charneau and Clavel, 1991; Charneau et al, 1992, 1994) that HIV1 and other lentiviruses have a more complex reverse transcription (RT°) strategy than oncoviruses whereby the presence of two additional cisacting sequences within the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS), leads to the formation of a threestranded DNA structure, the central DNA Flap. Mutations within the cPPT lead to a linear genome lacking the central DNA Flap, and severely impair viral replication (Zennou et al, 2000; Arhel et al, 2006a). Subcellular fractionation together with localization of viral DNA by fluorescence in situ hybridization (FISH) showed that central DNA Flapdefective molecules dock with wildtype kinetics at the nuclear membrane (Zennou et al, 2000). This docking, which involves microtubule followed by actindirected movements (Arhel et al, 2006b), is a very rapid process, as the vast majority of the linear DNA is already associated with the nuclear fraction within 6 h postinfection (p.i) or before (Barbosa et al, 1994; Zennou et al, 2000), as is confirmed by realtime imaging of HIV1 complexes in the cytoplasm of infected cells (McDonald et al, 2002; Arhel et al, 2006b). However, unlike wildtype viral DNA, Flapdefective linear DNA then accumulates at close proximity of the nuclear membrane, indicating a late defect in nuclear import (Zennou et al, 2000). The central DNA Flap therefore acts as a cisdeterminant of HIV1 genome nuclear import, and thus accounting, in part, for the mitosisindependent replication of lentiviruses. More precisely, studies of viral genome localization by in situ hybridization with electron microscopy indicated that this nuclear import defect occurs immediately before viral genome translocation through the nuclear pore and that most Flap defective DNA molecules have not initiated translocation through the nuclear pore (Arhel et al, 2006c).

Consistently with the cisacting role of the central DNA Flap, its reinsertion in HIV1derived gene transfer vectors complements the level of nuclear import from a strong defect to wildtype nuclear import levels, quantitatively indistinguishable from wildtype virus (Zennou et al, 2000). As a result, DNA Flapcontaining lentiviral vectors adhere closely to the early steps of wildtype virus infection. Reinsertion of the DNA Flap in HIV1 vectors strongly stimulates gene transfer efficiencies both in vivo and ex vivo in all tissue and cell types examined (see references in Arhel et al, 2006a), thus making the DNA Flap an essential and widely used component of lentiviral gene transfer vectors. It has been suggested that the role of the central DNA Flap, whereas beneficial in the context of HIV1derived vectors, could be nonessential for viral replication in the context of fulllength infectious viral genomes (Dvorin et al, 2002; Limon et al, 2002; Marsden and Zack, 2007). However, although mutations in the central DNA Flap do not lead to totally noninfectious viruses, thus pointing to a small fraction of nuclear import that is independent of the DNA Flap, we have recently shown that cPPT mutant viruses show an important nuclear import defect irrespective of the viral strain, cell type or infectivity assay used (Arhel et al, 2006a).

A threestranded DNA structure acting as a cisdeterminant of nuclear import is a novel and intriguing biological phenomenon with no known cellular or viral counterparts. In this work, we unmask the involvement of the central DNA Flap in an unexpected step of the HIV1 replication cycle: the maturation at the nuclear membrane of RT complexes (RTCs), consisting in a structure proceeding directly from the core of the particle, into preintegration complexes (PICs) of a size compatible with translocation through the nuclear pore. We further demonstrate that the routing of viral complexes to the nuclear membrane is independent of viral DNA synthesis. This work also revisits the identity of active intracellular HIV1 replication complexes: direct observation by scanning electron microscopy (SEM) indicates that uncoating, also called decapsidation, does not occur as an immediate postfusion event, but rather on the cytoplasmic side of the nuclear pore. Maturation of RTC into PIC is impaired in the absence of DNA Flap formation, therefore leading to the trapping of HIV1 PIC within an integral capsid (CA) shell prohibiting nuclear entry of the HIV1 genome.

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