HIV-1 must enter the nucleus and integrate its DNA into host genome for successful infection. However, the mechanism by which the viral complex docks at the nuclear envelope (NE) and enters the nucleus is not well understood. Although CA is known to play a critical role in nuclear import, the CA determinants that influence NE docking and viral complex translocation through the nuclear pore have not been defined. To identify the critical CA determinants, we developed quantitative live-cell imaging assays to study the NE docking and nuclear import of single viral complexes.
A high-throughput live-cell imaging assay was developed to study NE docking and residence times of single viral complexes labeled with either HIV-1 integrase-superfolder green fluorescent protein (sfGFP), APOBEC3F-yellow fluorescent protein (A3F-YFP) or Cyclophilin A-red fluorescent protein (CypA-DsRed). The amount of CA associated with viral complexes was quantified using a newly developed direct CA label (GFP-CA); CA was also detected by immunostaining with anti-CA antibody. Infectivity was determined in HeLa cells, and the CEM-SS and MT4 T cell lines.
Using high-throughput live-cell imaging, we identified CA mutants M10I, M10V and I15V that exhibited longer NE residence times compared to wild-type viral complexes in a CypA-dependent manner. Additionally, the M10 mutant complexes that entered the nucleus had longer NE residence times compared to WT, but only for the CypA-dependent nuclear import pathway. Analysis of virions labeled with CypA-DsRed also indicated that most viral complexes lost CypA-DsRed prior to nuclear import. CA mutants did not show infectivity defects in HeLa cells but were defective in T cell lines. Direct labeling of CA (GFP-CA) indicated that the CA levels of WT and mutant viral complexes were similar; however, CA detection using anti-CA antibody suggested differences in mutant viral complexes that reduced anti-CA antibody binding as a result of differences in conformation or host protein binding.
We have identified CA determinants that play a critical role in NE docking and nuclear import in a CypA-dependent manner. We propose a model in which CypA stabilizes the initial interaction of the viral core with NE but does not enter the nucleus with the viral core. These studies provide valuable insights into the interactions between the viral complex and the NE that result in stable docking and nuclear import.