The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.

We have generated a mouse where the clotting factor IX ( FIX ) gene has been disrupted by homologous recombination. The FIX nullizygous (−/−) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (−/−) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.

Regulation of HIV‐1 gene expression by the viral Tat trans ‐activator is a critical step in the viral life cycle. Tat acts as a highly unusual transcription factor that interacts with a stem‐loop RNA structure (TAR) found at the 5 end of all viral transcripts. There, it induces a modification of chromatin at the HIV‐1 long terminal repeat (LTR) promoter and stimulates the recruitment of elongationcompetent RNA polymerase II complexes capable of processive transcription. Increase of transcriptional elongation is the consequence of the interaction of Tat with cyclin T1, the cyclin component of CDK9, which phosphorylates the carboxy‐terminal domain of RNA polymerase II to enhance its processivity. Tat‐induced transcriptional activation of the LTR promoter is concomitant with recruitment of the transcriptional coactivators p300 and the highly homologue cAMP‐responsive transcription factor binding protein (CBP). These large proteins act at the level of transcriptional initiation by bridging the basal transcription machinery with specific transcriptional activators. Furthermore, p300/CBP are histone acetyl‐transferases capable of modulating the interaction of nucleosomes with DNA and with chromatin remodeling complexes. Besides histones, Tat itself is a substrate for the enzymatic activity of p300/CBP and of the associated factor P/CAF, suggesting a regulatory role of acetylation on the protein itself. Devising a unifying model for LTR activation that includes activities of Tat at the levels of both transcriptional initiation and transcriptional elongation is a challenging task at this moment. Nevertheless, protein localization studies indicate that both cyclin T1 and p300/CBP co‐localize in specific subnuclear compartments, thus suggesting participation of both proteins in the formation of multimolecular complexes governing coordinated steps of transcriptional activation.

Background: In living cells, proteins are in continuous motion and interaction with the surrounding medium and/or other proteins and ligands. These interactions are mediated by protein features such as electrostatic and lipophilic potentials. The availability of protein structures enables the study of their surfaces and surface characteristics, based on atomic contribution. Traditionally, these properties are calculated by physico-chemical programs and visualized as range of colors that vary according to the tool used and imposes the necessity of a legend to decrypt it. The use of color to encode both characteristics makes the simultaneous visualization almost impossible, requiring these features to be visualized in different images. In this work, we describe a novel and intuitive code for the simultaneous visualization of these properties. Methods: Recent advances in 3D animation and rendering software have not yet been exploited for the representation of biomolecules in an intuitive, animated form. For our purpose we use Blender, an open-source, free, cross-platform application used professionally for 3D work. On the basis Blender, we developed BioBlender, dedicated to biological work: elaboration of protein motion with simultaneous visualization of their chemical and physical features. Electrostatic and lipophilic potentials are calculated using physico-chemical software and scripts, organized and accessed through BioBlender interface. Results: A new visual code is introduced for molecular lipophilic potential: a range of optical features going from smooth-shiny for hydrophobic regions to rough-dull for hydrophilic ones. Electrostatic potential is represented as animated line particles that flow along field lines, proportional to the total charge of the protein. Conclusions: Our system permits visualization of molecular features and, in the case of moving proteins, their continuous perception, calculated for each conformation during motion. Using real world tactile/sight feelings, the nanoscale world of proteins becomes more understandable, familiar to our everyday life, making it easier to introduce "un-seen" phenomena (concepts) such as hydropathy or charges. Moreover, this representation contributes to gain insight into molecular functions by drawing viewer's attention to the most active regions of the protein. The program, available for Windows, Linux and MacOS, can be downloaded freely from the dedicated website http://www.bioblender.eu