Isolation of a peptide inhibitor of human rhinovirus.

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.

Genetic selection for modulators of a retinoic-acid-responsive reporter in human cells.

We used a genetic screening methodology, a human cell line bearing a retinoic-acid-responsive enhanced GFP reporter, and a flow sorter to recover dominant modulators of reporter expression. Four inducers and three suppressors that were fused to the C terminus of a protein scaffold for stability were isolated and their mechanisms of action studied. Mutagenesis experiments indicated that six of these dominant agents exerted their effects at the protein level. The single cDNA coding fragment that was isolated comprised the central 64-amino-acid section of human cyclophilin B, which contained its peptidyl-prolyl isomerase domain; this cyclophilin fragment repressed expression of the retinoic-acid-responsive reporter. The remaining clones encoded peptides shorter than 30 amino acids unrelated to known gene open reading frames. Genetic epistasis studies between the strongest inducer, R3, and a dominant-negative mutant of RARalpha suggest that the two factors function in the same pathway. Transcript microarray analyses suggest that R3 induced a subset of the retinoid-responsive genes in melanoma cells. Finally, yeast two-hybrid assays and co-immunoprecipitation studies of human cell extracts identified PAT1 as a protein that interacts with R3.

Renal principal cell-specific expression of green fluorescent protein in transgenic mice.

The purpose of this study is to develop transgenic mice with principal cell-specific expression of green fluorescent protein (GFP). After the cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of the promoter were used to drive expression of GFP in transgenic mice. In transgenic mice, GFP was selectively expressed in principal cells of the renal collecting duct and not in intercalated cells. Expression was increased by dehydration of mice. AQP2 and GFP expression was maintained in primary cultures of renal medulla that were stimulated with cAMP or vasopressin analogs. GFP-expressing cells were then isolated by fluorescence-activated cell sorting. RT-PCR analysis showed expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and cAMP response element binding protein but not H+-ATPase B1 subunit or anion exchanger 1. After expansion of these cells in culture, RT-PCR analysis showed continued expression of the same genes. This pattern of gene expression is that of principal cells rather than intercalated cells. This transgenic mouse model can be used in future studies of gene expression during the development, differentiation, and maturation of renal principal cells.

Stable expression of Anthozoa fluorescent proteins in mammalian cells.

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.