Isolation of 16L virus: a rapidly transforming sarcoma virus from an avian leukosis virus-induced sarcoma.

We have isolated a replication-defective rapidly transforming sarcoma virus (designated 16L virus) from a fibro-sarcoma in a chicken infected with td107A, a transformation-defective deletion mutant of subgroup A Schmidt-Ruppin Rous sarcoma virus. 16L virus transforms fibroblasts and causes sarcomas in infected chickens within 2 wk. Its genomic RNA is 6.0 kilobases and contains sequences homologous to the transforming gene (fps) of Fujinami sarcoma virus (FSV). RNase T1 oligonucleotide analysis shows that the 5' and 3' terminal sequences of 16L virus are indistinguishable from (and presumably derived from) td107A RNA. The central part of 16L viral RNA consists of fps-related sequences. These oligonucleotides fall into four classes: (i) oligonucleotides common to the putative transforming regions of FSV and another fps-containing avian sarcoma virus, UR1; (ii) an oligonucleotide also present in FSV but not in UR1; (iii) an oligonucleotide also present in UR1 but not in FSV; and (iv) an oligonucleotide not present in either FSV, UR1, or td107A. Cells infected with 16L virus synthesize a protein of Mr 142,000 that is immunoprecipitated with anti-gag antiserum. This protein has protein kinase activity. These results suggest that 16L virus arose by recombination between td107A and the cellular fps gene.     

Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.     

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.Antibodies have excellent target-binding specificities that have been exploited for the targeting of anticancer therapeutics specifically to tumor cells and the tumor microenvironment with success clinically (1). Recent technological advances have begun to improve the tumor targeting capabilities of antibodies and antibody-linked therapeutics by engineering antibodies with modified properties: molecular size [Fabs, single-chain Fv antibodies (scFvs), and single-domain], antigen-binding affinity, specificity, and valency (1–3). The optimal characteristics of particular antibodies and antibody fragments may vary considerably depending on the precise application. Therefore, technologies to efficiently optimize a lead antibody for a particular application are crucial for improving the efficacy of the therapeutic. As efficient rational design of antibodies is currently not feasible, the optimization of antibodies is best achieved by the randomization and subsequent selection of antibody mutants with the desired phenotypes by using polypeptide display technology.Display technology refers to methods of generating libraries of modularly coded biomolecules displayed usually on microorganisms (bacteriophage, bacteria, yeast) and screening them for particular properties (4, 5). The key feature of display technology is the linkage of a particular phenotype (displayed polypeptide) to its genotype (gene encoding the displayed polypeptide), enabling the rapid identification of the selected polypeptide(s). The most popular polypeptide display technology, phage display technology, has provided a versatile technology for the discovery and characterization of a variety of protein–protein interactions (6, 7). However, bacteriophage display has significant limitations, mainly related to the restrictions imparted by Escherichia coli on the expression, assembly, folding, transport, and posttranslational modifications of the viral protein fusions and their incorporation into viral particles (8). Finally, poor expression of the protein in eukaryotic cells can occur after selection using microorganism display platforms significantly delaying scale-up for therapeutic applications.A robust, eukaryotic version of bacteriophage display would offer a solution to this technology bottleneck, enabling an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to significantly improve promising antibody candidates compared with antibody display and affinity maturation using microorganisms. Recently, a mammalian cell surface antibody display system has offered one new approach, but this technology depends on the transient transfection of expression plasmids (9, 10). A eukaryotic display technology that also has a virus display platform as well as cell surface display capabilities would be ideal. A display platform based on eukaryotic retroviruses could offer a solution: replicating in eukaryotic cells with an efficient quality control system that assesses whether a protein has been properly folded and modified, including retroviral glycoproteins, before transport to the cell surface and incorporation into virions (11, 12). Previous published work demonstrated that the eukaryotic retrovirus MLV could function as a polypeptide display platform (13, 14). However, since those initial studies, characteristics that significantly limit the usefulness of MLV as a display platform have been found: significant shedding of the displayed polypeptides, the fact that certain displayed polypeptides could block MLV infection, and the fact that a significant reduction in MLV infectivity occurred when displaying viral glycoprotein–polypeptide fusions (15, 16).We have previously demonstrated the initial feasibility of a display platform based on another eukaryotic retrovirus, the avian leukosis virus (ALV), with the ability to display a wide range of polypeptide sizes including scFvs as fusions with ALV envelope glycoproteins (17), and with the ability to generate and screen a randomized uncensored peptide display library with >10⁶ diversity (18). This work demonstrated that the characteristics of ALV replication solved the severe limitations of polypeptide display using MLV, and provided a robust eukaryotic viral platform for the display of eukaryotic polypeptides as ALV surface (SU) glycoprotein fusions. The ability of the ALV genome to maintain at least 2.5 kb of additional sequence extends the possible sizes of the displayed polypeptides and/or offers space for an independent gene to encode a reporter protein or a second library of displayed polypeptides and still allow the RCAS series of replication-competent vectors to replicate to high titers in avian cells (19).In this study, we demonstrate that ALV display can be used to optimize ligand binding affinity as well as protein expression of a model scFv, providing a proof of principle that libraries of scFvs displayed as genetically stable ALV SU glycoprotein fusions offer a stable, soluble, relatively inert eukaryotic display platform for the display and selection of antibody libraries. Libraries of scFvs randomized at critical genomic sequence hotspots were generated and displayed as ALV SU glycoprotein fusions on virions, and then selected, improving the affinity of the model scFv more than 2,000-fold. The selection also significantly improved the expression level of the selected ALV SU-scFv fusion glycoproteins.     

Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene.

A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types. Each set contained wild-type and repair-deficient mutants. One set of cells contained a lacY mutation and the other a deletion of the entire lac operon. Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration. The results indicate that when no homologous sequences are present in the chromosomal DNA, mutations occur at a low frequency: at 10% survival the frequency was 1-2 X 10(-4) mutants per transformant. When homologous sequences, the lacY allele, are present in the chromosomal DNA, Lac- plasmids are found at a high frequency in a recA-dependent, lexA-independent fashion: at 10% survival the frequency was 5-10 X 10(-2) mutants per transformant. Southern blot analysis of the restriction enzyme profiles of the resulting plasmid and host-cell DNA sequences showed recombinational transfer of host sequences to the N-acetoxy-2-acetylamino-fluorene-treated plasmid had occurred. When the host chromosomes contained Lac+ homologous sequences no mutants were found, indicating that the results were not caused by error-prone recombination.     

DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid.

Ltk- aprt- mouse L cells were transformed to the tk+ phenotype with 10 ng of the herpes simplex virus-1 thymidine kinase (tk) gene and 20 micrograms of pBR322 or simian virus 40 (SV40) DNA. DNAs from five cloned cell lines show restriction endonuclease fragments that hybridize to both tk and pBR322 or SV40 DNA. In all of the cell lines some of these fragments also contain cellular DNA sequences. The use of carrier DNAs with defined sequences has enabled us to demonstrate that the joining of carrier and selectable gene sequences occurs in mouse cells. In one case we have been able to use the ampicillin resistance marker of pBR322 to "rescue" a recombinant plasmid. An analysis of the junction between pBR322 and tk in this plasmid suggests that a small area of homology (16 of 19 base pairs) might be involved in the recombination process.     

Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5'' long terminal repeat and the start of the gag gene.

The minimal cis-acting sequences outside the long terminal repeat (LTR) required for formation of an infectious retrovirus cloning vector were determined with recombinants of spleen necrosis virus (SNV) DNA and herpes simplex virus type 1 thymidine kinase gene. The 3' end of SNV DNA was removed to within 40 base pairs (bp) from the 3' LTR with only a 2-fold effect on the recovery of infectious recombinant virus. However, when the 5' end of SNV DNA was removed to within 100 bp from the 5' LTR, infectious recombinant virus was not recovered. Deletion mutants constructed around this latter region showed that nucleotides between 100 and 285 bp from the 5' LTR are necessary for encapsidation of genomic viral RNA. We call this region required for encapsidation E.     

Symmetrical base preferences surrounding HIV-1, avian sarcoma/leukosis virus, and murine leukemia virus integration sites

To investigate retroviral integration targeting on a nucleotide scale, we examined the base frequencies directly surrounding cloned in vivo HIV-1, murine leukemia virus, and avian sarcoma/leukosis virus integrations. Base preferences of up to 2-fold the expected frequencies were found for three viruses, representing P values down to <10(-100) and defining what appear to be preferred integration sequences. Offset symmetry reflecting the topology of the integration reaction was found for HIV-1 and avian sarcoma/leukosis virus but not murine leukemia virus, suggesting fundamental differences in the way different retroviral integration complexes interact with host-cell DNA.     

Independent regulation of endogenous and exogenous avian RNA tumor virus genes.

3H-Labeled complementary DNA specific for the envelope glycoprotein (env) gene of avian leukosis-sarcoma viruses was isolated by selective nucleic acid hybridization techniques, and used to analyze the expression of the endogenous provirus. The endogenous provirus in certain cell types termed chicken helper factor positive (chf+) can synthesize the envelope glycoprotein. Env DNA sequences were present in both chf+ and chf- cells, but env RNA was detectable only in positive cell types. When these cells were infected with the Bryan strain of Rous sarcoma virus (BH-RSV), a defective virus which is deleted in the env gene, the levels of endogenous env RNA remained unchanged, although exogenous BH-RSV specific RNA was synthesized in very high amounts. Thus, the infecting virus did not appear to influence the expression of the endogenous virus. Likewise, the endogenous virus did not influence the exogenous virus expression, since similar amounts of BH-RSV specific RNA were present in all infected cell types, regardless of the level of endogenous virus expression.     

Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates.

We have detected nucleotide sequences related to the transforming gene of avian sarcoma vius (ASV) in the DNA of uninfected vertebrates. Purified radioactive DNA (cDNAsarc) complementary to most of all of the gene (src) required for transformation of fibroblasts by ASV was annealed with DNA from a variety of normal species. Under conditions that facilitate pairing of partially matched nucleotide sequences (1.5 M NaCl, 59 degrees), cDNAsarc formed duplexes with chicken, human, calf, mouse, and salmon DNA but not with DNA from sea urchin, Drosophila, or Escherichia coli. The kinetics of duplex formation indicated that cDNAsarc was reacting with nucleotide sequences present in a single copy or at most a few copies per cell. In contrast to the preceding findings, nucleotide sequences complementary to the remainder of the ASV genome were observed only in chicken DNA. Thermal denaturation studies of the duplexes formed with cDNAsarc indicated a high degree of conservation of the nucleotide sequences related to src in vertebrate DNAs; the reductions in melting temperature suggested about 3--4% mismatching of cDNAsarc with chicken DNA and 8--10% mismatching of cDNAsarc with the other vertebrate DNAs.     

Cellular sequences are present in the presumptive avian myeloblastosis virus genome.

EcoRI restriction endonuclease fragments from a lambda proviral DNA hybrid containing the entire presumptive avian myeloblastosis virus (AMV) provirus, and from a lambda proviral hybrid containing a partial myeloblastosis-associated virus type 1 (MAV-1)-like provirus were compared by heteroduplex analysis. The cloned presumptive AMV provirus was also analyzed by electron microscopy, using R-loop formation with purified 35S RNA isolated from virions of the standard AMV complex. The results indicate that the putative AMV genome contains a segment absent in its MAV-1-like helper virus. This segment represents a substitution in the region of the genome that in MAV-1 virus is occupied by the envelope gene and is approximately 900 +/- 160 nucleotide pairs in length. Hybridization of specific probes from the presumptive AMV genome to Southern blots of EcoRI-digested cellular DNA has revealed that these substituted sequences are homologous to chicken and duck DNA that is not related to chicken endogenous proviral sequences.