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Cellular motor protein KIF-4 associates with retroviral Gag
1Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4-Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.
Insights
Cellular motor protein KIF-4 binds to human immunodeficiency virus type 1 (HIV-1) Gag proteins. This association suggests KIF-4 may be crucial for retroviral Gag protein transport within cells.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Retroviral Gag proteins are essential for assembling new virus particles.
- Kinesin superfamily motor protein 4 (KIF-4) is involved in intracellular transport.
Purpose of the Study:
- To investigate the association between KIF-4 and retroviral Gag proteins, particularly from human immunodeficiency virus type 1 (HIV-1).
- To identify the specific Gag protein forms and KIF-4 domains involved in this interaction.
Main Methods:
- Yeast two-hybrid assays to screen for protein interactions.
- Mammalian cell systems to confirm interactions.
- Biochemical fractionation techniques including sucrose gradient centrifugation, size-exclusion, and anion-exchange chromatography.
- Immunoprecipitation assays to verify protein binding.
Main Results:
- KIF-4 was found to associate with Gag proteins from multiple retroviruses, including HIV-1.
- Co-fractionation studies confirmed the association of KIF-4 with HIV-1 Gag in cellular extracts.
- KIF-4 primarily binds to the HIV-1 Gag precursor (Pr55 Gag) and a processing intermediate (Pr42), with binding mediated by a C-terminal KIF-4 domain.
Conclusions:
- The interaction between KIF-4 and HIV-1 Gag proteins is experimentally verified.
- KIF-4 may play a significant role in the intracellular transport of retroviral Gag proteins, potentially influencing virus assembly and maturation.