Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry (original) (raw)

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 .