Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.

In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a Michaelis-Menten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.

High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells.

Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.

Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line.

Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.

In situ window cleaning by laser blowoff through optical fiber.

The feasibility of a window cleaning system based on the laser blowoff technique is investigated to remove the impurity deposition on vacuum windows of the modified reversed field experiment fusion device. The laser pulse is sent to the window through a fused silica fiber optic (phi=1 mm), then focused on its internal surface, single shot ablating up to approximately 5 mm(2) of the impurity layer; the focused pulse is scanned across the window to clean its entire surface. The composition of the deposited layer is studied through the secondary ion mass spectrometry and profilometry techniques. Effectiveness of cleaning is analyzed in terms of quality of the cleaned spot, its dimension, repetition rate of the laser, and its wavelength. The energy damage threshold of the fiber optic is also investigated. Three different lasers (microjoule Nd:YAG, Nd:YLF, and ruby) are first tested directly on the window; then only the ruby laser beam is propagated through an optical fiber and tested.

Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion.

It has been proposed that membrane fusion events such as virus-cell fusion proceed through a hemifusion intermediate, a state where lipids but not contents of the fusing compartments mix. We engineered the influenza hemagglutinin (HA) such that it would be anchored in membranes via a glycosylphosphatidylinositol (GPI) tail. GPI-anchored HA forms a trimer that can bind red blood cells (RBCs) and change conformation under fusion-inducing conditions. Using RBCs labeled with fluorescent lipid or fluorescent soluble content probes, we found that GPI-anchored HA mediated lipid mixing with similar time course and efficiency as wt-HA, yet did not mediate transfer of soluble contents. Hence, GPI-anchored HA appears to initiate, but not complete, a fusion reaction. We interpret our results as evidence for uncoupling a physiological fusion reaction, for trapping a hemifusion intermediate, and for assigning a role to a transmembrane domain in a fusion event.

Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation.

To investigate the possible role of viral glycoprotein mobility in membrane fusion, fluorescence photobleaching recovery was employed to study the effects of exposure to mildly acidic pH (required to convert many viral fusion proteins to the fusion-active form) on the lateral mobility of influenza hemagglutinin (HA) proteins expressed at the surface of transfected cells. HA proteins from two different strains were compared: X:31 HA, which is activated by a brief exposure to pH 4.9 but is irreversibly inactivated at longer exposure times, and HA from A/Japan/305/57, which is relatively stable to inactivation at this pH [Puri, A., Booy, F.P., Doms, R.W., White, J.M., & Blumenthal, R. (1990) J. Virol. 64, 3824-3832]. The HA proteins from both strains, expressed in CV-1 cells using VS-40 vectors, exhibited relatively unrestricted lateral diffusion at the cell surface. The high mobility persisted following a brief exposure (1 min) to pH 4.9 to mediate conversion to the fusogenic state. Longer times (up to 15 min) of preincubation at pH 4.9 inhibited the lateral mobility of X:31 HA (the lateral diffusion rate was markedly reduced, followed by immobilization) but not of A/Japan HA, whose fusion activity is resistant to such treatment. Inhibition of the lateral mobility of X:31 HA due to preincubation at low pH was not specific to the CV-1 cells and was found also in a CHO cell line stably expressing this protein. The results presented demonstrate a close correlation between loss of mobility and inactivation of fusogenic activity, in accord with the notion that lateral motion of the HA proteins is required for fusion.(ABSTRACT TRUNCATED AT 250 WORDS)