Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases.

One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature.

Mechanistic analysis of pause site-dependent and -independent recombinogenic strand transfer from structurally diverse regions of the HIV genome.

Retroviral recombinants are generated by strand transfers occurring within internal regions of the viral genome and are a major source of genetic variability. Strand transfer has been linked to "pausing" occurring at secondary structures during synthesis by reverse transcriptase. Yet, weakly structured templates lacking strong pause sites also undergo efficient transfer. In this report, transfer crossover sites on high and low structured templates from the gag-pol frameshift region (GagPol) and the env (Env) regions, respectively, were determined by using a reconstituted in vitro strand transfer assay. The assay tested transfers occurring between a donor and acceptor template over a 150-nucleotide homologous region. The majority of crossovers were in a small 23-nucleotide region near a major pause site on GagPol, clearly indicating a pause-driven mechanism. In contrast, on Env, transfers were more dispersed clustering toward the end of the homologous region. Slowing down polymerization on Env by decreasing the dNTP concentration resulted in crossovers shifting toward the beginning of the homologous region. Removal of a small 38-nucleotide region at the 3'-end of the Env acceptor had a large effect on the level of strand transfer despite very few crossovers mapping to this region. This implicated this part of the acceptor in transfers occurring at downstream positions. For Env the results support a mechanism where the acceptor rapidly binds nascent DNA, then "zippers" downstream catching up with the donor-DNA hybrid and displacing the donor. Such a mechanism may be important to recombination in low structure regions of the HIV genome.

Differing roles of the N- and C-terminal zinc fingers in human immunodeficiency virus nucleocapsid protein-enhanced nucleic acid annealing.

The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing.

Evidence for the differential effects of nucleocapsid protein on strand transfer in various regions of the HIV genome.

An in vitro strand transfer assay that mimicked recombinational events occurring during reverse transcription in HIV-1 was used to assess the role of nucleocapsid protein (NC) in strand transfer. Strand transfer in highly structured nucleic acid species from the U3 3' long terminal repeats, gag-pol frameshift region, and Rev response element were strongly enhanced by NC. In contrast, weakly structured templates from the env and pol-vif regions transferred well without NC and showed lower enhancement. The lack of strong polymerase pause sites in the latter regions demonstrated that non-pause driven mechanisms could also promote transfer. Assays conducted using NC zinc finger mutants supported a differential role for the two fingers in strand transfer with finger 1 (N-terminal) being more important on highly structured RNAs. Overall this report suggests a role for structural intricacies of RNA templates in determining the extent of influence of NC on recombination and illustrates that strand transfer may occur by several different mechanisms depending on the structural nature of the RNA.