Intracellular Transport Of Pokeweed Antiviral Protein In Mammalian Cells

Pokeweed antiviral protein(PAP)is a typeâ… ribosome-inactivating protein produced by the pokeweed plant(Phytolacca americana).It is a multifunctional protein which has broad spectrum antiviral and anti-cancer antivities.Recent interest in PAP has been aroused by its inhibition of HIV-1 replication in infected cells. However,very little is known about the mechanism by which the typeâ… ribosome inactivating protein(RIP)such as PAP accesses its intracellular substrates in mammalian cells.By expressing the full length of the PAP gene with the pET-44a(+) system in E.coli BL21(DE3),and using an ELISA-based HIV-1 integrase assay,we demonstrated that PAP inhibited HIV-1 integrase in vitro.By visualizing the intracellular transport of PAP with immuno-gold labeling electro-microscopy,we showed that PAP reached both the endoplasmic reticulum and the nucleus of HEP-G2 cells.This importation of PAP into the nucleus may be important,since there it could cleave the viral genome or inhibit HIV-1 integration.1.Expression and purification of recombinant PAP.BL21(DE3)was transformed with plasmid pET-44a(+)-PAP using CaCl₂-mediated method.Expression was induced with 1 mM IPTG at 37℃for 3 h. Cells were then harvested by centrifugation.After sonication,the cell lysate was centrifuged at 12000 rpm for 15 min and the pellet containing the inclusion bodies was washed and then solubilized in denaturation buffer.After dialysis,the renatured protein was first purified through a column of Blue Sepharose CL-6B,and then the eluted protein was applied to a CM-Sepharose Fast Flow column for further purification.PAP purity was analyzed by 12%SDS-PAGE and the protein concentration was measured by the Bradford method.Cell viability was evaluated by the MTT assay and the IC₅₀value was calculated.The IC50 values of PAP for HeLa cells were 22μg/ml,and 17μg/ml for HEP-G2 cells.We used a highly purified PAP preparation to determine if PAP can act on supercoiled DNA.upon incubation with PAP,the supercoiled form of pBluescript SK was converted to the relaxed and linear forms in a concentration-dependent manner.These results indicated that the recombinant PAP recovered its biological activity2.Preparation of polyclonal antibody.Two adult male rabbits(New Zealand)were simultaneously immunized with a mixture of 1 mg purified PAP and the same volume of complete Freund's adjuvant. Every 2 weeks,0.5 mg of the antigen mixed with an equal volume of incomplete Freund's adjuvant were given subcutaneously a total of 4 times.Before immunization, blood samples were taken from the marginal vein of the rabbit ear and the sera were frozen as blank controls.The antiserum titer was measured by enzyme-linked immunoassay(ELISA)and the specificity was measured by western blot.Western blot showed that only the PAP was recognized by the antibody(Fig.5).The result indicated that the antiserum had high specificity for PAP.3.ELISA-based HIV-1 integrase assay.HIV-1 integrase is a key enzyme that integrate the viral gene into the human genome.To determine if PAP can inhibit the integration activities of HIV-1 integrase in vitro,we employed an non-radioactive ELISA-based assay and mimic the integration process in vivo.The result indicated that PAP can inhibit HIV-1 integrase in vitro.The concentration of PAP required for 50%inhibition of HIV-1 integrase was 1.82μg/ml. 4.Competitive ELISA experimentsThe two M13 phage clones displaying the heptamer peptides TPSHSSR and HPERATL were selected from a phage display library of randomized linear heptamer peptides by HIV-1 integrase in our laboratory.The heptamer peptides TPSHSSR and HPERATL are potential HIV-1 integrase inhibitors.The IC50 were 54.56±5.18μM and 28.292±1.32μM respectively.To determine if PAP can compete with these peptides in inhibiting HIV-1 integrase,we performed a competitive ELISA assay. HIV-1 integrase was coated on a microtitre plate at final concentration of 2μg/well. The HIV-1 integrase-bound plate was incubated with serial dilutions of PAP,and an adequate dilution of the M13 phage clone displaying the peptide TPSHSSR.or HPERATL for 1 h at room temperature.Bound phage was revealed using an anti-M13 horseradish peroxidase-labeled antibody(1:5000)detected with TMB.The result showed that PAP was able to competitively inhibit the binding of TPSHSSR to HIV-1 integrase in a dose-dependent manner,while there was almost no inhibition of the clone displaying HPERATL(Fig.7),suggesting that PAP and the TPSHSSR heptamer may have the same binding site on HIV-1 integrase.5.Localization of endocytosed PAP in HEP-G2 cells by immuno-gold labeling electro-microscopy.This assay was performed according to our previous report with some modifications.Briefly,HEP-G2 cells were treated with 375μg/ml PAP and PBS(as control)for 3.5 h at 37℃.After endocytosis,the cells were harvested and encapsulated.Sections were cut with a glass knife in a cryo-ultramicrotome at about -110℃.The ultrathin cryosections were treated with rabbit anti-PAp antibody(1:500 dilution)at 4℃overnight.After washing 6 times with label buffer,the sections were incubated with 15 nm colloidal gold-Affinipure goat anti-rabbit IgG(1:20 dilution). Colloidal gold accumulated in the ER and nucleus of PAP-treated HEP-G2 cells,while no gold particles were present in the ER and nucleus of PBS-treated cells(Fig.8).This result gave direct evidence that PAP reached the ER and nucleus of HEP-G2 cells.