Comparison Between Several Integrase-defective Lentiviral Vectors Reveals Increased Integration of an HIV Vector Bearing a D167H Mutant (original) (raw)
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Mutations affecting interaction of integrase with TNPO3 do not prevent HIV-1 cDNA nuclear import
Retrovirology, 2011
Background: Integration of human immunodeficiency virus type 1 (HIV-1) into a host cell chromosome is an essential step under the control of the viral integrase (IN). Although this enzyme is necessary and sufficient to catalyze the integration reaction in vitro, cellular cofactors are involved in the process in vivo. The chromatinassociated factor LEDGF/p75 interacts with IN and promotes integration to transcription units of the host genome. HIV-1 IN also binds the karyopherin TNPO3, however the significance of this interaction during viral replication remains to be explored. Results: Here we present a functional analysis of IN mutants impaired for LEDGF/p75 and TNPO3 interaction. Among them, IN W131A and IN Q168L, that were previously identified to be deficient for LEDGF/p75 interaction, were also partially impaired for TNPO3 binding. We observed that mutations abolishing IN ability to form tetramers resulted in a severe reduction in LEDGF/p75 binding. In sharp contrast, no correlation could be found between the ability of IN to multimerize and TNPO3 interaction. Most of the mutant viruses were essentially impaired for the integration step whereas the amount of 2-LTR circles, reflecting the nuclear import of the viral DNA, was not significantly affected.
Proceedings of the National Academy of Sciences, 2006
Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262 RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. . These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.
Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry
Scientific Reports, 2016
Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies. Although active nuclear import is a hallmark in the replication cycle of lentiviruses such as the human immunodeficiency virus type 1 (HIV-1), nuclear entry is one of the least understood steps 1-4. After reverse transcription of the viral RNA into double stranded DNA, the pre-integration complex (PIC) is formed as an assembly of the viral DNA (vDNA) and cellular and viral proteins. Prior to integration, the PIC has to cross the natural barrier of the nuclear membrane through nuclear pore complexes (NPCs) which serve as selective entry gates 5. Recent evidence suggests that uncoating of the HIV capsid (CA) core occurs close to the nuclear membrane although some CA molecules may accompany the PIC into the nucleus 6-9. Genome-wide siRNA screens identified the nucleoporins Nup153 and Nup358 (RANBP2) as host cofactors of HIV nuclear import 10-13. Nup358 binds CA 14 and is believed to act as a docking station for the HIV PIC 10,14. Nup153 is located in the nuclear basket; interactions between its FG repeats and either viral integrase (IN) or CA are in line with a role during nuclear entry 10,15,16. Besides nucleoporins, importin α /β , importin 7 and Transportin-SR2 (TRN-SR2, TNPO3) have been proposed to be involved in nuclear import of the PIC 1,17-20. A role for the HIV DNA flap in nuclear import has been proposed as well 21,22. HIV-1 IN mediates the insertion of the viral cDNA in two consecutive steps: 3′ processing and strand transfer 23. IN catalytic activity is highly dependent on a dynamic equilibrium of IN multimers; evidence indicates that 3′ processing requires at least a dimer whereas at least a tetramer is needed for concerted integration 24-28. In line with this, the prototype foamy virus (PFV) intasome has been shown to consist of an IN tetramer 29. Concerted integration of the HIV cDNA occurs into active transcription sites 30,31 and is guided by the host factor LEDGF/p75 32-34. LEDGF/p75 contains an N-terminal chromatin/DNA binding moiety (residues 1-325) and a C-terminal integrase binding domain (IBD, residues 347-429) 35,36. The pivotal role of LEDGF/p75 in HIV-1 replication was revealed via mutagenesis, RNAi-mediated depletion, transdominant overexpression of the IBD of LEDGF/p75 and cellular knockout studies 32,33,37-43 .
High-level expression of active HIV-1 integrase from a synthetic gene in human cells
The FASEB Journal, 2000
A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.-Cherepanov, P., Pluymers, W., Claeys, A., Proost, P., De Clercq, E., Debyser, Z. High-level expression of active HIV-1 integrase from a synthetic gene in human cells.
Virology Journal, 2010
Background During the early stage of HIV-1 replication, integrase (IN) plays important roles at several steps, including reverse transcription, viral DNA nuclear import, targeting viral DNA to host chromatin and integration. Previous studies have demonstrated that HIV-1 IN interacts with a cellular Lens epithelium-derived growth factor (LEDGF/p75) and that this viral/cellular interaction plays an important role for tethering HIV-1 preintegration complexes (PICs) to transcriptionally active units of host chromatin. Meanwhile, other studies have revealed that the efficient knockdown and/or knockout of LEDGF/p75 could not abolish HIV infection, suggesting a LEDGF/p75-independent action of IN for viral DNA chromatin targeting and integration, even though the underlying mechanism(s) is not fully understood. Results In this study, we performed site-directed mutagenic analysis at the C-terminal region of the IN catalytic core domain responsible for IN/chromatin binding and IN/LEDGF/p75 int...
Journal of Virology, 2004
Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegra...
Journal of Virology, 2006
After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three-to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two-to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly-and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.
Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells
Biochemical and Biophysical Research Communications, 2005
Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration. We have previously selected stable cell lines (293T-IN S ) that constitutively express high levels of HIV-1 integrase (IN) from a synthetic gene [FASEB J. 14 (2000) 1389]. We have now constructed linear DNA substrates containing the terminal HIV LTR sequences (so called Ômini-HIVÕ) and EGFP as reporter gene to evaluate whether IN can improve the integration of transfected linear DNA. After electroporation of this mini-HIV we observed a 2-to 3-fold increase in EGFP expression in IN expressing cell lines relative to control cells. The increase in EGFP expression was still evident after passaging of the cells. The effect was observed with linear DNA but not with circular DNA, thus excluding an effect on DNA uptake. The increase was the highest in the 293T-IN S (D64V) cell line due to an increase in the amount of total mini-HIV DNA and 2-LTR circles as quantified by Q-PCR. Our data suggest that IN over-expressed in our cell lines interacts with the incoming DNA, protects it from nuclease degradation but does not catalyze the integration as such.