Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.     

Microphotometry of rat liver nucleoproteins during the cell cycle, and comparison of diploid nuclei in the G2 period with tetraploid nuclei.

The following facts were established with a microphotometric investigation of isolated nuclei from rat liver in different stages of the cell cycle. During the mitotic wave occurring in the liver of newborn animals after injection of casein it was found that the naphtol yellow S (NYS) protein content of the nuclei increases about 30% during the G1-period. A second increase of around 70% was established during the S-phase whereas no increase could be observed during the G2-phase. An indication for the existence of a "critical protein mass" of the nuclei before the onset of the S-phase could be observed. The protein content of diploid nuclei in the G1-phase of adult animals is about 50% higher than in newborns. This makes it probable that there is no significant difference in the NYS-protein content of diploid nuclei in the G2-period and tetraploid nuclei of adult rats. No differences were observed between diploid nuclei in the G2-period of newborn rats and tetraploid nuclei of adult rats in their Fastgreen histon, RNA and SH plus SS content. The only criterion to distinguish between G2 nuclei and tetraploid nuclei seems to be the number of nucleoli, but this is rather unreliable.     

B cells regulate murine gammaherpesvirus 68 latency

The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (gammaHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating gammaHV68 in vitro) and limiting dilution PCR (frequency of cells carrying gammaHV68 genome) assays to compare gammaHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent gammaHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying gammaHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated gammaHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying gammaHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated gammaHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic gammaHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of gammaHV68 latency, (ii) there are organ-specific differences in the efficiency of gammaHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of gammaHV68 from latency, and (iv) B cells are important for controlling chronic gammaHV68 infection.     

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02 µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (V_mem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only V_mem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and V_mem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.     

The role of beta2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.