C-terminal deletion mutant of pokeweed antiviral protein inhibits viral infection but does not depurinate host ribosomes.
Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. In addition to its ribosome-inactivating ability, PAP has potent antiviral activity against many plant and animal viruses, including HIV. We recently described the isolation and characterization of nontoxic PAP mutants, NT123-2, which has a point mutation (E176V) in the active site that abolishes enzymatic activity, and NT124-3, which has a nonsense mutation that results in deletion of the C-terminal 25 aa (W237Stop). In vitro translation of rabbit reticulocyte lysate ribosomes was inhibited by the C-terminal deletion mutant, but not by the active site mutant. We expressed both mutants in transgenic tobacco and showed that, unlike PAP or variant PAP, neither mutant is toxic to transgenic plants. In vivo depurination of rRNA was detected in transgenic tobacco expressing variant PAP, but not in transgenic plants expressing either the active site mutant or the C-terminal deletion mutant PAP. When extracts from transgenic plants containing the mutant PAPs were exogenously applied to tobacco leaves in the presence of potato virus X (PVX), the C-terminal deletion mutant had antiviral activity, while the active site mutant had no antiviral activity. Furthermore, transgenic plants expressing low levels of the C-terminal deletion mutant showed resistance to PVX infection, while transgenic plants expressing very high levels of the active site mutant PAP were not resistant to PVX. Our results demonstrate that an intact active site of PAP is necessary for antiviral activity, toxicity, and in vivo depurination of tobacco ribosomes. However, an intact active site is not sufficient for all these activities. An intact C terminus is also required for toxicity and depurination of tobacco ribosomes in vivo, but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity.
Tumer
N E
NE
Agricultural Biotechnology Center and Department of Plant Pathology, Rutgers University, Cook College, New Brunswick, NJ 08903-0231, USA. Tumer@aesop.rutgers.edu
Hwang
D J
DJ
Bonness
M
M
D000818:Animals; D017353:Gene Deletion; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000821:virology; D017863:Potexvirus; D018749:RNA, Plant / Q000235:genetics*; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000235:genetics*
D010940:Plant Proteins; D018749:RNA, Plant; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein