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

Abstract

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...

Regulation of Ribosomal Proteins on Viral Infection

“…PAP serves as effective inhibitor of many animal and plant viruses, for instance, HIV-1, poliovirus, HSV-1, TMV and Tobacco etch virus, etc. [128][129][130]. The antiviral activity of PAP has been explored by many researchers, via conjugating a variety of monoclonal antibodies for antiviral therapeutics [131,132] and generating transgenic plants resistant to plant viruses [133].…”

Limitations of the Human‐PBL‐SCID Mouse Model for Vaginal Transmission of HIV‐1

“…Standard amplification (nucleic acid sequence‐based amplification [NASBA]) and detection assay was performed according to the manufacturer's recommendations. Detection was based on electrochemiluminescence signals 11,33 …”

“…Unlike macaque/SIV or SHIV non‐human primate models, which are not widely available, need special housing and handling in addition to slow breeding rates, the rodent model with large cohort sizes to carry a large number of experiments with shorter time‐period and reproducibility permit statistical evaluation of antiviral therapies against HIV‐1 11,32,33 . An advantage of this in vivo system is that it recapitulates the host immune environment at the time of infection and favors the transmission of CCR5‐using viruses, as is observed clinically 34 .…”

Radiolabeled Antibodies for Therapy of Infectious Diseases

“…HIV infection of hu-PBL SCID mice led to the loss of CD4+ T cells, as it is also seen as a result of HIV infection in human hosts. Mosier model has been broadly utilized in evaluating efficacy of anti-viral drugs [29, 30]. Similar to the splenic model, RIT led to a several log reduction in viral load in groups treated with 25, 50 and 100 µCi 213 Bi-2556 relative to controls (P<0.05).…”