Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55Gag molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55 Gag . The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.
Elastin is a critical component of the lung interstitium, providing the property of recoil to the vascular, conducting airway, and terminal airspace compartments of the lung. Elastic fibers, consisting of soluble tropoelastin monomers cross-linked on a preexisting scaffold of microfibrils, are produced primarily during late fetal and neonatal stages of development. The factors and molecular mechanisms regulating the cell type-specific and tightly temporally regulated expression of tropoelastin are currently under investigation. The onset and inductive phase of tropoelastin expression are characterized by increased transcription of the tropoelastin gene. Glucocorticoids accelerate this induction in fetal rats during the canalicular stage of lung development. Many additional factors regulate tropoelastin expression in cultured lung fibroblasts and vascular smooth muscle cells, but the in vivo roles of such mediators are still under investigation. Cell-cell interactions may also promote elastogenesis during lung development, as localization of tropoelastin mRNA in pseudo-glandular and canalicular lungs demonstrates a close spatial relationship between epithelium and adjacent elastogenic mesenchyme. Elastin metabolism is altered in several experimental models of bronchopulmonary dysplasia, characterized by abnormal lung morphological development, suggesting that normal elastin production and deposition is necessary for proper development of alveoli. Studies employing reverse genetics may prove useful in further defining the role of elastin in lung development.
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