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C Kempf

Publications and source records attributed to C Kempf.

At least 37 records · Page 2Linked to original sources
PubMed1996

The fragmentation of incoming Semliki Forest virus nucleocapsids in mosquito (Aedes albopictus) cells might be coupled to virion uncoating.

The fate of Semliki Forest virus (SFV) nucleocapsid, especially the capsid protein (C-protein), was investigated during the early stages of a productive infection in mosquito Aedes albopictus cells. Infection of the cells resulted in a time dependent accumulation of a C-protein derived fragment. This fragmentation of incoming viral nucleocapsid was prevented by NH4Cl, an agent generally used to elevate the pH in acidic intracellular compartments, suggesting that a low intravesicular pH is required for this process. Density gradient analysis of the postnuclear cell lysate demonstrated that the fragmentation was associated with a cellular compartment showing a density of 1.14 +/- 0.02 g/ml. This cellular compartment was devoid from a lysosomal marker enzyme and represented the timely preceding cellular fraction through which SFV passed before encountering a lysosomal fraction. Furthermore, the intracellular distribution of the viral, 3H-uridine-labeled RNA suggested that the same fraction might represent a key cellular compartment in which the separation of the viral RNA from the viral structural proteins is primed. In conclusion, these data lead to the suggestion that the fragmentation of incoming SFV nucleocapsids in Aedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection.

Aedes↗
PubMed1996

Virus inactivation by pepsin treatment at pH 4 of IgG solutions: factors affecting the rate of virus inactivation.

BACKGROUND: IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material. STUDY DESIGN AND METHODS: The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content. RESULTS: Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. In contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus. CONCLUSION: Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.

Animals↗
PubMed1996

Low pH-induced pore formation by spike proteins of enveloped viruses.

Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.

Aedes↗
PubMed1995

Identification of the pore forming element of Semliki Forest virus spikes.

Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.

Aedes↗
PubMed1994

Modulation of thermoprotection and translational thermotolerance induced by Semliki Forest virus capsid protein.

Low amounts of Semliki Forest virus capsid protein transferred into target cells by electroporation-mediated delivery (10(3)-10(4) molecules incorporated/cell) confer thermal resistance resulting in enhanced survival. Furthermore, when exposed to 43 degrees C, these cells display an enhanced expression of heat-shock protein-70 and a translational thermotolerance. Similarly, low amounts of capsid protein transferred into cells in which transcription is blocked by actinomycin D, also protect the translational machinery at 43 degrees C. In a cell-free translation system, added capsid protein appears to modulate translational efficiency of endogenous mRNAs. At approximately 1 molecule/ribosome, capsid protein is able to enhance translation at 30 degrees C and at 43 degrees C. In contrast, high concentrations of capsid protein are responsible for a marked inhibition of protein synthesis at 30 degrees C, but only hamper translational thermotolerance at 43 degrees C. Our results favor the hypothesis that small amounts of capsid protein trigger a chaperone-like activity that is able to protect the translational machinery from thermal damage.

Adaptation, Biological↗
PubMed1993

pH-dependent pore formation in Semliki forest virus-infected Aedes albopictus cells.

The plasma membrane properties of Semliki forest virus-infected Aedes cells were studied using whole-cell patch-clamp recording. Cells exhibited a marked increase in membrane conductance, gm (from 0.48 +/- 0.09 nS to 14.2 +/- 10.8 nS) upon exposure to acidic pH (5.6). The membrane potential depolarized from -40.9 +/- 5.2 mV to -2.43 +/- 7.14 mV under these conditions. In uninfected cells, there was no change in gm after lowering the pH. This implies that viral fusion proteins are involved in changing the membrane properties at low pH. The increased gm in infected cells at low pH was not persistent but declined within minutes. Millimolar concentrations of calcium and zinc prevented or reverted the increase in gm. The results suggest that viral proteins, if brought to their fusogenic conformation by exposure to low pH, form unspecific pores in the residing membrane. These pores might play a role in virus entry.

Aedes↗
PubMed1993

Early events of Semliki Forest virus-induced cell-cell fusion.

Insect cells (Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, Ij. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, Ij increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in Ij are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of Ij steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., Ij steps occurred within less than 1 msec. The absence of Ij flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.

Aedes↗
PubMed1993

Semliki Forest virus core protein fragmentation: its possible role in nucleocapsid disassembly.

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243-255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.

Aedes↗
PubMed1993

A comparative study of the effect of dextran sulfate on the fusion and the in vitro replication of influenza A and B, Semliki Forest, vesicular stomatitis, rabies, Sendai, and mumps virus.

The effect of dextran sulfate on the fusion of a series of enveloped viruses, bearing specifically different fusion proteins, was investigated. The fusion with model- and with biological membranes was monitored by an R18 fluorescence-dequenching fusion assay. Dextran sulfate strongly suppresses the fusion of orthomxyo- (influenza A (H1N1 and H3N2 subtypes) and influenza B), of toga- (Semliki Forest virus), and of rhabdoviruses (vesicular stomatitis and rabies virus). The fusion of the paramyxo-viruses Sendai and mumps was not significantly affected by the anionic polysaccharide. The response to dextran sulfate was virus-specific, and identical for the different members of one virusfamily, bearing the same fusion protein. It was shown that dextran sulfate attaches with high affinity to the viruses studied, but not to erythrocytes. The anionic polymer appears to attach to the fusion epitope of the viral membrane. The inhibition of virus replication in vitro shows a remarkable correlation with the observed anti-fusion effects of dextran sulfate.

Dextran Sulfate↗
PubMed1993

Arachidonic acid and other unsaturated fatty acids alter membrane potential in PC12 and bovine adrenal chromaffin cells.

The action of arachidonic acid and other fatty acids on membrane potential in PC12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1-40 microM) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K(+)-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC12 and bovine chromaffin cells by modulating K+ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca2+ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca2+ influx and secretion. We propose a model where internally generated fatty acids act as a feedback to desensitize the stimulated cell via inhibition of receptor-dependent Ca2+ influx and induction of membrane hyperpolarization.

Animals↗
PubMed1992

Changes in membrane permeability during Semliki Forest virus induced cell fusion.

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (less than 6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influx of protons into the cells (Kempf et al. Biosci. Rep. 7, 761-769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (M(r) less than 1000) was observed when infected cells were exposed to a pH less than 6.2. The pH dependence of the permeability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.

Aedes↗
PubMed1992

Cytoplasmic bridges and gap junctions in an insect cell line (Aedes albopictus).

Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used study simultaneously the diffusional and electrical properties of intercellular junctions. Diffusion studies involved injection of fluorescent molecules into one cell of a cell pair and visual inspection of their intercellular redistribution. Electrical measurements involved a dual voltage clamp method and whole-cell recording with patch pipette. The voltage clamp protocol was aimed at examining the dependency of the junctional conductance, gj, on membrane potential, Vm. Cell pairs exhibiting a voltage-dependent gj were found to allow intercellular diffusion of Lucifer Yellow CH (molecular mass, 443 Da), but not of FITC-dextran (molecular mass, 4,400 Da). This response pattern is consistent with the presence of gap junctions in the intercellular junctions. Cell pairs showing no voltage dependence of gj were found to permit intercellular diffusion of both Lucifer Yellow CH and FITC-dextran (dextran labelled with fluorescein isothiocyanate). This behaviour is compatible with the presence of cytoplasmic bridges connecting the two adjacent cells. Hence, in culture the cells investigated express two kinds of intercellular structures, gap junctions and cytoplasmic bridges.

Aedes↗
PubMed1992

Electrical coupling between cells of the insect Aedes albopictus.

1. Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used to study the electrical properties of intercellular junctions. A double voltage-clamp approach was adopted to control the voltage gradient between the cells and measure the intracellular current flow. 2. Determinations of junctional conductance (gj) revealed two types of intercellular contacts, gap junctions and cytoplasmic bridges. Identification occurred by means of functional criteria, i.e. the dependency of gj on (i) junctional membrane potential, (ii) non-junctional membrane potential, and (iii) heptanol. 3. In cell pairs with putative gap junctions, gj was dependent on the junctional membrane potential (Vj). When determined at the beginning of voltage pulses, gj was insensitive to Vj; when determined at the end of 15 s pulses, it depended on Vj in a bell-shaped manner (70% decrease for a change in Vj of +/- 75 mV). 4. These cell pairs also showed a dependency of gj on the non-junctional membrane potential (Vm). When determined immediately after changing the non-junctional membrane potential in both cells, gj was not affected by Vm; when determined 30 s later, gj was modified by Vm in a S-shaped fashion (100% decrease when Vm was depolarized to +50 mV). 5. Exposure to 3 mM-heptanol gave rise to complete and reversible block of gj in cell pairs with putative gap junctions. 6. Cell pairs susceptible to uncoupling by heptanol revealed junctional currents indicative of the operation of gap junction channels. The single-channel conductance, determined at a Vm of -50 to -70 mV, was 133 pS. 7. In the case of putative cytoplasmic bridges, gj was insensitive to the junctional and non-junctional membrane potential. In addition, it was not affected by 3 mM-heptanol. 8. While most cell pairs showed functional properties characteristic of gap junctions or cytoplasmic bridges, few cell pairs exhibited junctional currents compatible with the co-existence of both junctional structures.

Aedes↗
PubMed1991

Semliki Forest virus envelope proteins function as proton channels.

It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH exposure (Soederlund et al., 1972). This contraction of the nucleocapsids has been used as an indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain digestion, which were used as a control, did not translocate protons. This implies that the ectodomain of the spike plays a crucial role for the proton translocation.

Aedes↗
PubMed1991

Virus inactivation during production of intravenous immunoglobulin.

The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families--human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)--were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.

Animals↗
PubMed1990

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH.

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (less than 6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.

Aedes↗