Pokeweed Antiviral Protein Isoforms PAP-I, PAP-II, and PAP-III Depurinate RNA of Human Immunodeficiency Virus (HIV)-1

Rajamohan, Francis; Venkatachalam, T. K.; Irvin, James D.; Uckun, Fatih M.

doi:10.1006/bbrc.1999.0922

Cited by 82 publications

(66 citation statements)

References 14 publications

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“…Aniline Cleavage Assays of Ribosomal RNA Depurination-5 g of E. coli 16 S and 23 S rRNA (Roche Molecular Biochemicals) or 30 g of total ribosome prepared from the rabbit reticulocyte-rich whole blood (25) were incubated with either 5 or 25 g of wild-type or mutant rPAP proteins in 50 l (final volume) of binding buffer (25 mM Tris⅐HCl, pH 7.8, 10 mM KCl, 5 mM MgCl 2 , 2% glycerol) at 37°C for 1 h. The RNA was extracted with phenol:chloroform (24:24), precipitated with ethanol, and treated with 20 l of 1 M aniline acetate (pH 4.5) for 30 min on ice. The RNA was precipitated with ethanol, electrophoresed in a 6% urea/ polyacrylamide gel, and stained with ethidium bromide as described previously (22).…”

Section: Bacterial Strains and Construction Of Mutants-recombinant Wimentioning

confidence: 99%

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

Rajamohan¹,

Pugmire²,

Kurinov³

et al. 2000

Journal of Biological Chemistry

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The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAPmediated depurination of ribosomal RNA.

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Section: Bacterial Strains and Construction Of Mutants-recombinant Wimentioning

confidence: 99%

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

Rajamohan¹,

Pugmire²,

Kurinov³

et al. 2000

Journal of Biological Chemistry

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“…The release of adenine or guanine from E. coli 23S and 16S rRNAs (Roche Molecular Biochemicals) and HIV-1 RNA (ABI Biotechnologies, Columbia, Md.) was measured with an HPLC system (Hewlett-Packard Co., Palo Alto, Calif.) equipped with a diode array detector and a ChemStation software program for data analysis, as described previously (18,20). Briefly, 2 g of the RNA substrate was incubated with 2.5 M wild-type or mutant PAPs for 1 h at 37°C in 50 l of binding buffer (25 mM Tris-HCl [pH 7.8], 10 mM KCl, 5 mM MgCl 2 , 2% glycerol).…”

Section: Methodsmentioning

confidence: 99%

“…PAP has a unique ability to depurinate human immunodeficiency virus (HIV) type 1 (HIV-1) RNA (18,20). PAP exhibits potent antiviral activity against nucleoside analog reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV-1 isolates (3).…”

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confidence: 99%

Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

Uckun

Rajamohan

Pendergrass

et al. 2003

Antimicrob Agents Chemother

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Pokeweed antiviral protein (PAP) is a 29-kDa naturally occurring antiviral agent that can be isolated from the leaves of the pokeweed plant (Phytolacca americana) (6, 36). PAP has a unique ability to depurinate human immunodeficiency virus (HIV) type 1 (HIV-1) RNA (18,20). PAP exhibits potent antiviral activity against nucleoside analog reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV-1 isolates (3). Both zidovudine (ZDV)-sensitive and ZDV-resistant clinical HIV-1 isolates were found to be Ͼ4 logs more sensitive to PAP than to ZDV (3). We have cloned the gene for PAP and established procedures for the large-scale production and purification of the cloned recombinant PAP (16,17). We have tested recombinant PAP against a broad panel of viruses in vitro and documented that it is as active as native PAP against both DNA and RNA viruses (16,17). We were also able to determine the X-ray crystal structure of PAP at a 2.1-Å resolution (8, 9).More recently, we have used a molecular model of PAP-RNA interactions (19,21,22) for the rational design of PAP mutants with potent anti-HIV activities. In the present study, two such recombinant PAPs, FLP-102 ( 151 AA 152 ) and ), have been engineered, produced, and tested both in vitro and in vivo. These proteins depurinate HIV-1 RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. Our preliminary studies indicate that these proteins are substantially less toxic than wild-type PAP and exhibit potent in vivo activities against a genotypically and phenotypically NRTI-resistant clinical HIV-1 isolate in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. We hypothesize that FLP-102 and FLP-105, because of their potent anti-HIV activities and lack of systemic toxicities, may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. MATERIALS AND METHODSEngineering of recombinant PAPs. Molecular modeling studies for the rational design of recombinant PAPs were performed as previously described in detail (19,21,22). Recombinant wild-type PAP (phosphate-buffered saline [PBS]-PAP) was obtained by subcloning the PAP-I gene (amino acids 22 to 313) into the pBluescript SKϪ expression vector (17). An uracil-containing template of PAP was obtained by transforming Escherichia coli CJ236 with recombinant plasmid PBS-PAP. The oligonucleotides used for site-directed mutagenesis were synthesized on a 200-nmol scale and purified by high-pressure liquid chromatography (HPLC) by Biosynthesis Inc. (Lewisville, Tex.). The site-directed mutagenesis procedure was performed by using the Mutagene M13 in vitro mutagenesis kit (Bio-Rad, Hercules, Calif.), as described in the manufacturer's manual. DNA sequencing was carried out by the method of Sanger et al. (26) according to the instructions of the manufacturer (U.S. Biochemical Corp., Cleveland, Ohio). Fine chemicals and restriction enzymes were purchased from Roche Molecular Biochemicals (In...

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“…The pokeweed plant produces several isozymes of PAP, all exerting potent antiviral properties [11,13,[36][37][38][39][40][41][42]. PAP isozymes evoke depurination of genomic HIV-1 RNA [43][44][45], TMV RNA [46], poliovirus [47], herpes simplex virus (HSV) [48], influenza virus [49], and brome mosaic virus (BMV) [50], among many others, showing a broad spectrum of antiviral activity [13]. This depurination is concentration dependent.…”

Section: Activities Attributed To Papmentioning

confidence: 99%

Insights into the Molecular Antiviral Mechanism of Pokeweed Protein from Phytolacca americana

Aitbakieva¹,

Domashevskiy²

2016

Biochem Pharmacol (Los Angel)

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Pokeweed Antiviral Protein Isoforms PAP-I, PAP-II, and PAP-III Depurinate RNA of Human Immunodeficiency Virus (HIV)-1

Cited by 82 publications

References 14 publications

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

Insights into the Molecular Antiviral Mechanism of Pokeweed Protein from Phytolacca americana

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