A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template.
Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.
Hudak
K A
KA
Biotechnology Center for Agriculture and the Environment, and the Department of Plant Pathology, Cook College, Rutgers University, New Brunswick, New Jersey 08903, USA.
Wang
P
P
Tumer
N E
NE
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D002474:Cell-Free System / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010947:Plants, Toxic; D014176:Protein Biosynthesis / Q000502:physiology*; D011687:Purines / Q000737:chemistry / Q000378:metabolism*; D012315:RNA Caps; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic; D014026:Nicotiana / Q000737:chemistry
D000998:Antiviral Agents; D010940:Plant Proteins; D011687:Purines; D012315:RNA Caps; D012333:RNA, Messenger; D018749:RNA, Plant; D012367:RNA, Viral; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein