P39.09
Background: Vpx, a virus associated accessory protein is encoded by Human Immunodeficiency Virus type 2 (HIV-2) and Simian Immunodeficiency Virus (SIVsm/SIVmac lineage) is known to be involved in the nuclear import of viral DNA in non-dividing primary target. Interestingly, Vpx mutant virus fails to replicate in non-dividing cells. The mechanism by which Vpx helps in the nuclear transport of viral genome remains unknown.
Methods: Co-immunoprecipitation was performed to study the protein-protein interaction in transiently transfected HEK cells. Protein co-localization was assessed by immunofluoresence. Using homology modeling of Vpx and Nup153 amino acid sequences, putative binding motif for Vpx in Nup153 was predicted. Site directed mutagenesis was employed to generate mutant viruses which were defective for interaction with Nup 153. Nuclear import ability of wild type and mutant virus was analysed by 2-LTR assay.
Results: Molecular transport across nuclear envelope is governed by nucleo pore complexes (NPCs), composed of 30 nucleoporins (NUPs). We found that interaction between Vpx and human Nup153 was necessary for viral DNA import. We mapped the domain of Nup153 critical for interaction with Vpx and our data suggests the role of a zinc finger domain (610-869aa) to be critical. Vpx interaction with Nup153 was impaired by exchange of serine (63,65) and tyrosine (66, 69 and 71) residues to alanine and resulted in abrogation of nuclear localization. Interestingly, the SIV PBj1.9 with mutant Vpx was found to have reduced nuclear import ability in 2-LTR assay.
Conclusions: Our data gives insight into the mechanism of nuclear import by Vpx interaction with Nup153. Novel treatment methods using phosphorylation inhibitors specific for Vpx could be devised in the future.
