Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity,protein processing,and genomic RNA dimer stability

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.     

Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22 degrees C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found that cold-induced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-C18), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV) composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based of the similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.     

A RhoA-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3

The fusion glycoproteins of human respiratory syncytial virus (RSV) and human parainfluenza virus type-3 (PIV-3) mediate virus entry and syncytium formation. Interaction between the fusion protein of RSV and RhoA, a small GTPase, facilitates virus-induced syncytium formation. We show here a RhoA-derived peptide inhibits RSV and syncytium formation induced by RSV and PIV-3, both in vitro by inhibition of cell-to-cell fusion and in vivo by reduction of peak titer by 2 log10 in RSV-infected mice. These findings indicate that the interaction between these two paramyxovirus fusion proteins and RhoA is an important target for new antiviral strategies.     

Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies.

The effect of the carbohydrates trehalose, glucose, and hydroxyethyl starch (HES) on the motional properties of the phosphate headgroup of freeze-dried dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by means of 31P NMR, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results show that trehalose, which is a strong glass former (Tg = 115 degreesC), elevates the onset of the lipid headgroup rotations and preserves some rotational mobility of the phosphate headgroups after cooling from the liquid-crystalline state. Glucose (Tg = 30 degreesC), a very effective depressant of the phase transition temperature of freeze-dried DPPC, markedly elevates the initiation of the temperature of headgroup rotations. On the other hand, the monosaccharide does not preserve the headgroup disordering when cooled from the liquid-crystalline state. These effects are consistent with formation of hydrogen bonds between the OH groups of the sugar and the polar headgroups of DPPC. They show, however, that hydrogen bonding is not sufficient for preservation of the dynamic properties of freeze-dried DPPC. HES, although a very good glass former (Tg > 110 degreesC), does not depress the phase transition temperature and affects only slightly the rotational properties of freeze-dried DPPC. This lack of effect of HES is associated with the absence of direct interactions with the lipid phosphates, as evidenced by the FTIR results. These data show that vitrification of the additive is not sufficient to affect the dynamic properties of dried DPPC.