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
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 mitosis‐independent lentiviral replication.
We have previously shown (Charneau and Clavel, 1991; Charneau et al, 1992, 1994) that HIV‐1 and other lentiviruses have a more complex reverse transcription (RT°) strategy than oncoviruses whereby the presence of two additional cis‐acting sequences within the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS), leads to the formation of a three‐stranded 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 Flap‐defective molecules dock with wild‐type kinetics at the nuclear membrane (Zennou et al, 2000). This docking, which involves microtubule‐ followed by actin‐directed 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 post‐infection (p.i) or before (Barbosa et al, 1994; Zennou et al, 2000), as is confirmed by real‐time imaging of HIV‐1 complexes in the cytoplasm of infected cells (McDonald et al, 2002; Arhel et al, 2006b). However, unlike wild‐type viral DNA, Flap‐defective 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 cis‐determinant of HIV‐1 genome nuclear import, and thus accounting, in part, for the mitosis‐independent 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 cis‐acting role of the central DNA Flap, its reinsertion in HIV‐1‐derived gene transfer vectors complements the level of nuclear import from a strong defect to wild‐type nuclear import levels, quantitatively indistinguishable from wild‐type virus (Zennou et al, 2000). As a result, DNA Flap‐containing lentiviral vectors adhere closely to the early steps of wild‐type virus infection. Reinsertion of the DNA Flap in HIV‐1 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 HIV‐1‐derived vectors, could be nonessential for viral replication in the context of full‐length 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 three‐stranded DNA structure acting as a cis‐determinant 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 HIV‐1 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 pre‐integration 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 HIV‐1 replication complexes: direct observation by scanning electron microscopy (SEM) indicates that uncoating, also called decapsidation, does not occur as an immediate post‐fusion 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 HIV‐1 PIC within an integral capsid (CA) shell prohibiting nuclear entry of the HIV‐1 genome.
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