Maize Ribosome-inactivating Protein as an HIV-specific Cytotoxin

Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases which cleave the N-glycosidic bond of adenine-4324 at the alpha-sarcin/ricin (SR) loop of 28S rRNA. The depurination of the SR loop results in the inhibition of protein synthesis by impairing the binding of EF-1 or EF-2 to ribosomes. RIPs are therefore highly cytotoxic and have been used as abortificiant, anti-cancer and anti-HIV agents, either alone or as a component of immunotoxins. Many type I and II RIPs, such as MAP30, GAP30, DAP30, pokeweed antiviral protein (PAP) and ricin, have been reported to possess anti-HIV activity by inhibiting viral replication in vitro and in vivo though the anti-HIV mechanism is still unclear.;Maize RIP is classified as a type III RIP. It is synthesized in the endosperm of maize as an inactive precursor (Pro-RIP), which contains a 25-amino acid internal inactivation region. During germination, a two-chain activated form (MOD) is generated by endogenous proteolysis of the internal inactivation region, whereas the two chains (16.5 and 8.5 kDa) are tightly associated without disulfide linkage. Our group has solved the crystal structures of both the Pro-RIP and MOD and found that this internal inactivation region is on the surface of the N-terminal domain in Pro-RIP. The removal of this internal inactivation region increases the inhibition of protein synthesis of rabbit reticulocyte lysate by over 600 folds. The presence of the internal inactivation region has led us to derive a novel strategy to enhance the specificity of maize RIP towards HIV-infected cells while minimizing its cytotoxic effect on normal cells.;In this study, we provide an account on the generation of HIV-1 protease-sensitive maize RIP variants by first incorporating the HIV-1 protease recognition sequences to the internal inactivation region of the Pro-RIP. Among the five variants, three variants were cleaved and activated by HIV-1 protease in vitro and in vivo, resulting in an active two-chain form with N-glycosidase activity comparable to the fully active maize RIP. In addition, the variants inhibited viral replication in human T lymphocytes (C8166) infected by two T-tropic HIV-1 strains, HIV-1IIIB and HIV-1 RF/V82F/I84V, and their cytotoxicity towards uninfected cells was similar to the non-activated precursor (TAT-Pro). In comparison to TAT-Pro, variants TAT-Pro-HIV-MA/CA and Pro-TAT-Pro-HIV-p2/NC had 2- to 70-fold increase in the inhibition of p24 antigen production in the HIV-infected cells with low cytotoxicity towards uninfected C8166 cells.;In the future, the 25 as internal loop region of Pro-RIP can be modified for the optimized recognition of proteases of other HIV strains. This approach opens a new opportunity for the anti-HIV application of maize RIP and other related type III RIPs. A modified maize RIP may also be applied to target other viruses and pathogens, for examples, hepatitis C and malaria, which are dependent on pathogen-encoded proteases for replication.