Deguanylation of human immunodeficiency virus (HIV-1) RNA by recombinant pokeweed antiviral protein.
Modeling studies, combined with the molecular docking of the trinucleotide GGG into the active site of the deadenylating RNA N-glycosidase pokeweed antiviral protein (PAP), indicated that a guanine base can fit into the active site pocket of PAP without disturbing its unique geometry and is sandwiched between residues Tyr(72) and Tyr(123) very much like an adenine base. The guanine base can form two specific hydrogen bonds with the active site residues Ser(121) and Val(73) and the attached negatively charged phosphate groups can entertain stabilizing electrostatic interactions with two clusters of positively charged patches on the PAP surface formed by Lys(210) and Arg(179) from one side and Arg(122) and Arg(135) from the other side of the active site. These observations prompted the hypothesis that the RNA depurinating activity of PAP may not be restricted to adenine residues and PAP should be capable of deguanylating ribosomal and viral RNA as well. This hypothesis was experimentally confirmed by direct demonstration that guanine base is released from both ribosomal and HIV-1 RNA after treatment with purified recombinant PAP using quantitative high performance liquid chromatography. Recombinant PAP released adenine and guanine residues at a 1:1 ratio from HIV-1 RNA and at an approximately 3:1 (adenine:guanine) ratio from Escherichia coli ribosomal RNA. At a concentration of 5 microM, recombinant PAP released 263 +/- 10 pmol of adenine and 100 +/- 11 pmol of guanine from 1 microgram of E. coli ribosomal RNA (16S + 23S) within 4 h of treatment. By comparison, 138 +/- 12 pmol of adenine and 143 +/- 10 pmol of guanine were released from 1 microgram of HIV-1 RNA under identical treatment conditions (5 microM recombinant PAP, 4 h treatment). The deguanylation of the ribosomal and viral RNA targets by recombinant PAP was concentration-dependent and is abolished by alanine substitutions of the catalytic active site residues Tyr(72) and Tyr(123). To our knowledge, these findings provide the first evidence that PAP can deguanylate both ribosomal and viral RNA.
Rajamohan
F
F
Biotherapy Program, Hughes Institute, St. Paul, Minnesota, 55113, USA.
Kurinov
I V
IV
Venkatachalam
T K
TK
Uckun
F M
FM
D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1
D000227:Adenine Nucleotides; D000998:Antiviral Agents; D006150:Guanine Nucleotides; D009841:Oligonucleotides; D010940:Plant Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012367:RNA, Viral; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; C024275:pokeweed antiviral protein