Nuclear Localization of Human Immunodeficiency Virus Type 1 Preintegration Complexes (PICs): V165A and R166A Are Pleiotropic Integrase Mutants Primarily Defective for Integration, Not PIC Nuclear Import

Abstract

Retroviral replication requires the accomplishment of certain key steps in the early phase of the viral life cycle. Soon after entering a cell, the viral enzyme reverse transcriptase (RT) copies genomic RNA into linear double-stranded cDNA. The viral enzyme integrase (IN) then inserts this DNA into a host cell chromosome. In vivo, reverse transcription and integration take place in the context of large nucleoprotein complexes that are called reverse transcription complexes (RTCs) and preintegration complexes (PICs), respectively (reviewed in reference 29).Two different IN activities are required for integration. During an initial 3Ј processing reaction, IN cleaves each cDNA end adjacent to the phylogenetically conserved sequence CA. For both Moloney murine leukemia virus (MoMLV) (7,28,45) and human immunodeficiency virus type 1 (HIV-1) (12, 13, 39), 3Ј processing can occur in the cell cytoplasm. Following nuclear entry, IN transfers the processed 3Ј ends to the 5Ј phosphates of a double-stranded staggered cut in chromosomal DNA (7, 28). The final step of the integration process, which involves repairing the gaps between the unjoined viral 5Ј ends and the chromosome, can be completed by host cell enzymes (4, 54).The double lipid bilayer that envelops animal cell nuclei presents a formidable barrier for retroviral PICs. The nuclear envelope contains numerous nuclear pore complexes (NPCs) that permit the passive diffusion of relatively small macromolecules with diameters up to about 9 nm, which corresponds roughly to a 40-to 60-kDa globular protein (reviewed in reference 37). The relatively large size of retroviral PICs, estimated to be roughly the size of a ribosome for HIV-1 (25), precludes their passive transport through intact NPCs (reviewed in reference 27). Different retroviruses have apparently evolved different strategies to access the cell nucleus. HIV-1, for example, can be transported by an energy-dependent process in nondividing cells (9), suggesting that HIV-1 PICs contain specific nuclear localization sequences (NLSs) that govern their transport through intact NPCs (reviewed in references 27 and 52). Productive infection by MoMLV, in contrast, requires cells to pass through the M phase of the cell cycle (36, 44). Since animal cell nuclei disassemble during mitosis, MoMLV apparently reaches cell chromosomes in the absence of active nuclear transport by waiting for the dissolution of nuclear membranes.Recently, replication-defective MoMLV (55) and HIV-1 (3, 57) mutants were described as blocked at the nuclear import step in rapidly dividing cells. This suggested that retroviruses

Integrase Mutants Defective for Interaction with LEDGF/p75 Are Impairedin Chromosome Tethering and HIV-1Replication

“…Because HIV Q168A made twice as much total HIV cDNA as the WT virus, it was also 2-fold defective for two-LTR circles. The catalytic mutant D116A accumulated about 5 times more two-LTR circles than the WT virus, as described previously (31). Finally, by quantifying integrated proviruses at 24 and 48 h post-infection, both HIV Q168L and HIV Q168A mutants were deficient for integration much like the D116A mutant (Fig.…”

“…V165A, another IN mutant, was also shown to be defective for LEDGF/p75 interaction (9). A virus harboring the V165A mutation was replicationdeficient (31). Analysis of the IN structure shows that residues Val 165 and Gln 168 are in close contact within the monomer.…”

Integrase, LEDGF/p75 and HIV replication

“…This now legendary problem remains unsolved [23,124]. Despite the initially provocative correlation between intracellular trafficking of integrase proteins expressed outside the viral context and the differential capability of these retroviruses for non-dividing cell infection -HIV IN is nuclear, MLV IN is cytoplasmic -the weight of current evidence tilts against integrase or the integrase-p75 interaction being the determinant [75,95,96,101,121,[128][129][130][131][132][133]. As is discussed below, however, a role for p75 in PIC nuclear import is not definitively excluded.…”

Macrophages and HIV-1: dangerous liaisons

“…HIV integrase (IN) and matrix (MA) proteins carry sequences that may serve as nuclear localisation signals (NLS) for the recognition and targeting of the PIC to the nuclear pore by the importin-/ pathway (Gallay et al, 1997;Gallay et al, 1996). However, IN NLS mutants cannot replicate in either dividing and non-dividing cells, suggesting that IN NLS is involved in steps of replication other than nuclear import (Limon et al, 2002a;Petit et al, 2000;Tsurutani et al, 2000). IN has intrinsic karyophilic properties and may play a key role in nuclear import, independent of NLS (Bouyac-Bertoia et al, 2001;Depienne et al, 2001;Devroe et al, 2003).…”