The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal f luid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of f luorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potentialsensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal f luid.

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

BackgroundPhotochemical tissue bonding (PTB) is a promising sutureless technique for tissue repair. PTB is often achieved by applying a solution of rose bengal (RB) between two tissue edges, which are irradiated by a green laser to crosslink collagen fibers with minimal heat production. In this study, RB has been incorporated in chitosan films to create a novel tissue adhesive that is laser-activated.MethodsAdhesive films, based on chitosan and containing ~0.1 wt% RB were manufactured and bonded to calf intestine by a solid state laser (λ = 532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, tested the bonding strength. K-type thermocouples recorded the temperature (T) at the adhesive-tissue interface during laser irradiation. Human fibroblasts were also seeded on the adhesive and cultured for 48 hours to assess cell growth.ResultsThe RB-chitosan adhesive bonded firmly to the intestine with adhesion strength of 15 ± 2 kPa, (n = 31). The adhesion strength dropped to 0.5 ± 0.1 (n = 8) kPa when the laser was not applied to the adhesive. The average temperature of the adhesive increased from 26°C to 32°C during laser exposure. Fibroblasts grew confluent on the adhesive without morphological changes.ConclusionA new biocompatible chitosan adhesive has been developed that bonds photochemically to tissue with minimal temperature increase.

The transmembrane (TM) glycoprotein gp41 of human immunodeficiency virus type 1 possesses an unusually long (ϳ150 amino acids) and highly conserved cytoplasmic region. Previous studies in which this cytoplasmic tail had been deleted partially or entirely have suggested that it is important for virus infectivity and incorporation of the gp120-gp41 glycoprotein complex into virions. To determine which regions of the conserved C-terminal domains are important for glycoprotein incorporation and infectivity, several small deletions and amino acid substitutions which modify highly conserved motifs were constructed in the infectious proviral background of NL4.3. The effects of these mutations on infectivity and glycoprotein incorporation into virions produced from transfected 293-T cells and infected H9 and CEM؋174 cells were determined. With the exception of a mutation deleting amino acids QGL, all of the constructs resulted in decreased infectivity of the progeny virus both in a single-round infectivity assay and in a multiple-infection assay in H9 and CEM؋174 cells. For most mutations, the decreased infectivity was correlated with a decreased incorporation of glycoprotein into virions. Substitution of the arginines (residues 839 and 846) with glutamates also reduced infectivity, but without a noticeable decrease in the amount of glycoprotein incorporated into virus produced from infected T cells. These results demonstrate that minor alterations in the conserved C-terminal region of the gp41 cytoplasmic tail can result in reductions in infectivity that correlate for most but not all constructs with a decrease in glycoprotein incorporation. Observed cell-dependent differences suggest the involvement of cellular factors in regulating glycoprotein incorporation and infectivity.

Protein arginine methylation is a common post-translational modification where a methyl group is added onto arginine residues of a protein to alter detection by its binding partners or regulate its activity. It is known to be involved in many biological processes, such as regulation of signal transduction, transcription, facilitation of protein–protein interactions, RNA splicing and transport. The enzymes responsible for arginine methylation, protein arginine methyltransferases (PRMTs), have been shown to methylate or associate with important regulatory proteins of the cell cycle and DNA damage repair pathways, such as cyclin D1, p53, p21 and the retinoblastoma protein. Overexpression of PRMTs resulting in aberrant methylation patterns in cancers often correlates with poor recovery prognosis. This indicates that protein arginine methylation is also an important regulator of the cell cycle, and consequently a target for cancer regulation. The effect of protein arginine methylation on the cell cycle and how this emerging key player of cell cycle regulation may be used in therapeutic strategies for cancer are the focus of this review.