Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.

Purification, characterization and cytotoxic properties of a bacterial RNase.

An RNase produced by Bacillus safensis RB-5 was purified up to 22.32-fold by successive techniques of salting out, DEAE-anion exchange and gel permeation (Sephadex G-100) chromatography techniques with a yield of 2.27%. The purified RNase possessed a single band in SDS-PAGE (Mr ~ 60 kDa). The purified RNase showed optimal activity at temperature of 37 °C and pH 7.5 in the presence of substrate (Yeast RNA) and Mg2+ ions. The RNase activity was strongly inhibited by Hg2+ and mildly by Fe2+, Ba2+ and Zn2+ ions. Its half-life was found to be 8 h at 37 °C. The RNase kinetics study showed Km and Vmax value of 0.3 mM and 9.2 µmol/mg/min, respectively. The purified RNase also showed cytotoxic and antiproliferative activities towards a few transformed cell lines. The purified RNase (IC50 0.035 U/mL) effectively inhibited RD and Hep-2C cells proliferation & migration, while sparing HEK 293 cells. The purified RNase was cytotoxic as well as effective degrader of the RNA of transformed RD cells at low concentration. Moreover, the purified RNase of B. safensis RB-5 was found to possess a little hemolytic activity towards human RBCs.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

Fungal Ribotoxins: A Review of Potential Biotechnological Applications.

Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.

Purification and characterization of an extracellular ribonuclease from a Bacillus sp. RNS3 (KX966412).

Ribonucleases (RNases) catalyze the degradation of ribonucleic acid (RNA) into smaller nucleotides. RNases display angiogenic, neurotoxic, antitumor and immunosuppressive properties. In the present study, an extracellular RNase was successfully purified to homogeneity from a Bacillus sp. RNS3 (KX966412) by salting out at 0-50% ammonium sulphate saturation followed by the gel permeation (Sephadex G-100) chromatography. The multistep purification resulted in 10.4 fold purification of RNase with a yield of 3.12%. The activity of the purified RNase was found to be 2.02U/mg protein. The purified RNase was monomeric with a molecular weight of 66kDa. It exhibited Michalis-Menten kinetics parameters Kcat 7.92min-1 and Km 0.12mg/mL. The antiproliferative activity of the purified RNase was tested against an established Hep-2C (HeLa derived) cancer cell line in vitro. The purified RNase reduced the viability of the Hep-2C cells significantly with an IC50 value of 3.53µg/mL. The haemolytic activity of purified RNase was also evaluated and unfortunately, it showed a strong haemolytic activity towards human RBCs.

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin.

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against ribosomes and suggested as potential insecticides. Here we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are of interest.

[Cytotoxicity mechanism of the RNase Sa cationic mutants involves inhibition of potassium current through Ca2+-activated channels].

Bacterial ribonucleases (RNases) are considered to be potential anticancer agents. One of most important determinants of RNase cytotoxicity is the net charge of the molecule. In this work a set of mutants of the RNase from Streptomyces aureofaciens (RNase Sa), differing in the net charge of the protein molecules (from -7 to +6) and localization of additional positive charge at the N- or C-terminus of the molecule is used to study inhibition of cell growth. It has been found that the mutants of RNase with increased cationicity most effectively inhibit the growth of HEKhSK4 cells. Additional positive charge at the C-terminus of the molecule also increases the cytotoxic properties of RNases. It has been shown that RNase cytotoxicity correlated with the level of inhibition of the K+-current in cells.

Anticodon nuclease encoding virus-like elements in yeast.

A variety of yeast species are known to host systems of cytoplasmic linear dsDNA molecules that establish replication and transcription independent of the nucleus via self-encoded enzymes that are phylogenetically related to those encoded by true infective viruses. Such yeast virus-like elements (VLE) fall into two categories: autonomous VLEs encode all the essential functions for their inheritance, and additional, dependent VLEs, which may encode a toxin-antitoxin system, generally referred to as killer toxin and immunity. In the two cases studied in depth, killer toxin action relies on chitin binding and hydrophobic domains, together allowing a separate toxic subunit to sneak into the target cell. Mechanistically, the latter sabotages codon-anticodon interaction by endonucleolytic cleavage of specific tRNAs 3' of the wobble nucleotide. This primary action provokes a number of downstream effects, including DNA damage accumulation, which contribute to the cell-killing efficiency and highlight the importance of proper transcript decoding capacity for other cellular processes than translation itself. Since wobble uridine modifications are crucial for efficient anticodon nuclease (ACNase) action of yeast killer toxins, the latter are valuable tools for the characterization of a surprisingly complex network regulating the addition of wobble base modifications in tRNA.

Sustained and cancer cell targeted cytosolic delivery of Onconase results in potent antitumor effects.

The unfavorable pharmacokinetics and low tumor specificity hampered the potential clinical utility of Onconase, a promising modality in anticancer treatment with unique targets and novel mechanism of action. In this study, a modular and multi-stage drug delivery system (DDS) that can break down organ (renal accumulation), cellular (cancer cell specific uptake) and sub-cellular (endosomal escape) level barriers encountered by Onconase during its long journey from injection site to the cytoplasm of cancer cell was designed. Human serum albumin fusion extended the half-life of Onconase and significantly decreased its kidney accumulation. Epithelial cell adhesion molecular (EpCAM) specific antibody fragment appending enhanced binding and internalization of Onconase toward EpCAM positive cancer cell and increased its tumor accumulation and retention. Tethering Onconase to its carrier by cleavable disulfide linker prompted endosomal escape and restored its cytotoxicity. In vivo antitumor efficacy assay in human tumor xenograft model revealed that only when the entire organ, cellular and sub-cellular level barriers had been broken down, will Onconase turn into a potent antitumor agent.

A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A.

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.

Viral genome RNA degradation by sequence-selective, nucleic-acid hydrolyzing antibody inhibits the replication of influenza H9N2 virus without significant cytotoxicity to host cells.

Influenza A virus infection is a great threat to avian species and humans. Targeting viral proteins by antibody has a limited success due to the antigen drift and shift. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a sequence-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)-encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18-nucleotide single stranded nucleic acid substrate, dubbed asNP(18), the sequence of which is unique to the NP-vRNA. We isolated a 3D8 VL variant, NP25, that had ∼15-fold higher affinity (∼54nM) and ∼3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP(18)-selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any significant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP(18) sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a new strategy of targeting viral genomic RNA for degradation by antibody for the prevention of influenza virus infection in humans and animals.

RNA toxins: mediators of stress adaptation and pathogen defense.

RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

Eosinophil ribonucleases and their cutaneous lesion-forming activity.

Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to pathophysiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at >or=2.5 microM that began at 2 days, peaking at approximately 7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 microM) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosinophil-associated diseases.

Local controlled delivery of anti-neoplastic RNAse to the brain.

PURPOSE: Antineoplastic RNAse proteins, also known as Amphibinases, have been shown effective against various solid tumors but were found selectively neurotoxic to Purkinje cells in the cerebellum. This work describes the use of a waxy biodegradable poly(ricinoleic-co-sebacic acid) for the local controlled delivery of cytotoxic amphibinases in the parietal lobe of the brain in an attempt to overcome cerebellar neuronal toxicity while affecting glioma cells. METHODS: Amphibinase analogues were encapsulated in poly(ricinoleic-co-sebacic acid) formulations using mix-melt technology and loaded onto surgical foam. In-vitro release was monitored by BCA colorimetry and by RNAse specific bioactivity. The implants were inserted into rat brains bearing 9L glioma to assess toxicity and efficacy. RESULTS: The various formulations showed extended linear release for several weeks with minimal burst effect. Best in-vivo efficacy was obtained with ACC7201 containing implants, resulting in the extension of the median survival from 13 to 18 days with 13% long-term survivors. CONCLUSION: Antineoplastic proteins were released from a p(SA-RA) polyanhydride implants in a controlled manner, providing efficacy against 9L glioma, while evading neurotoxicity in the cerebellum. The controlled release of Amphibinases forms the potential for a new therapy against brain tumors.

The therapeutic potential of fungal ribotoxins.

Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of high-resolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the mid-term future.

Ranpirnase (Onconase), a cytotoxic amphibian ribonuclease, manipulates tumour physiological parameters as a selective killer and a potential enhancer for chemotherapy and radiation in cancer therapy.

BACKGROUND: Ranpirnase, a cytotoxic amphibian ribonuclease, is effective against cancer cells, inducing apoptosis independently of p53 protein. Onconase (the smallest member of the RNase A superfamily) has moved into clinical testing in the US and Europe. OBJECTIVE: The main focuses of this review are to examine the manipulation of tumour physiological parameters by ranpirnase and discuss its molecular, pharmacological and physiological roles in preclinical and clinical trials in terms of benefits and toxicity. METHODS: Relevant literature, including the author's unpublished presentations at recent conferences, was examined. RESULTS/CONCLUSION: In animal studies, improvements in tumour physiology (i.e., increased blood flow, inhibited oxygen consumption, increased oxygenation and decreased tumour hypertension) and selectively enhanced radiation responses (i.e., increased radiation sensitivity and inhibited repair of sublethal and potentially lethal damage) were observed after ranpirnase treatment in preclinical tumour models. Ranpirnase is a promising candidate as an enhancer for radiation- and chemotherapy. Ongoing clinical trials promise to further improve the treatment of mesothelioma and lung cancer.

Structural basis for catalysis by onconase.

Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5'-AMP complex at 1.65 A resolution and a wild-type ONC-d(AUGA) complex at 1.90 A resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k(cat)/K(M) profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10(2)-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k(cat)/K(M) value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes.

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.

Effect of wheat leaf ribonuclease on tumor cells and tissues.

The antiproliferative and antitumor effect of wheat leaf ribonuclease was tested in vitro on the human ML-2 cell line and in vivo on athymic nude mice bearing human melanoma tumors. The antiproliferative activity of this plant ribonuclease was negligible in comparison with bovine seminal ribonuclease. In the experiments in vivo, a significant decrease of the tumor size, however, was observed in the mice treated with wheat leaf ribonuclease (27 kDa) compared with the control RNase A and polyethylene glycol. In nude mice injected intratumoraly with wheat leaf ribonuclease, the tumor size decreased from 100% in the control mice to 39% in treated mice. In the mice treated with polyethylene glycol-conjugated wheat leaf ribonuclease, the tumor reduction was observed from 100 to 28%, whereas in counterparts treated with polyethylene glycol-conjugated bovine seminal ribonuclease the tumor inhibition was reduced from 100 to 33%. Certain aspermatogenic and embryotoxic activity of wheat leaf ribonuclease and bovine seminal ribonuclease also appeared, but was lower in comparison with the effect of onconase. Mutual immunological cross-reactivity between wheat leaf ribonuclease antigens on one side and animal RNases (bovine seminal ribonuclease, RNase A, human HP-RNase and onconase) on the other side proved a certain structural similarity between animal and plant ribonucleases. Immunogenicity of wheat leaf ribonuclease was weaker in comparison with bovine seminal ribonuclease (titer of antibodies 160-320 against 1280-2560 in bovine seminal ribonuclease). Interestingly, immunosuppressive effect of wheat leaf ribonuclease tested on mixed lymphocyte culture-stimulated human lymphocytes reached the same level as that of bovine seminal RNase. The antibodies against wheat leaf ribonuclease produced in the injected mice did not inactivate the biological effect of this plant RNase in vivo. This is probably the first paper in which plant ribonuclease was used as antiproliferative and antitumor drug against animal and human normal and tumor cells and tissues in comparison with animal ribonucleases.

Construction and use of retroviral vectors encoding the toxic gene barnase.

Suicide genes for negative selection of cells have been powerful tools in somatic cell genetic studies and in gene therapy. Here we report on the construction, characterization, and utilization of retroviral vectors encoding barnase, a ribonuclease from Bacillus amyloliquefaciens, expression of which results in apoptosis of transduced mammalian cells. High-titer viral vector production was enabled by expression of an inhibitor of barnase (barstar) in transfected cells generating murine leukemia virus (MLV)- and HIV-1-based vectors. To identify cellular genes required for infection we used barnase-encoding vectors in a genetic screen to isolate mutant mammalian cells that are resistant to infection by MLV and HIV-1. We describe one such mutant clone that is inhibited in the infection process after reverse transcription. These results suggest that barnase-encoding vectors should be useful for negative selection strategies examining retroviral infection from entry to integration. Furthermore these vectors could have utility in approaches for gene therapy that require specific cell ablation.