Biomedical subjects

A T Da Poian

Publications and source records attributed to A T Da Poian.

10 recordsLinked to original sources

PubMed2005

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Animals↗

PubMed2001

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization.

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.

Anilino Naphthalenesulfonates↗

PubMed1998

Kinetics of intracellular viral disassembly and processing probed by Bodipy fluorescence dequenching.

A novel method is described for the study of viral disassembly and processing in live cells. Vesicular stomatitis virus (VSV) was labelled with the fluorescent probe Bodipy-FL and the resulting conjugate was 97.6% self-quenched due to fluorescence resonance energy transfer between neighbouring Bodipy molecules. In vitro experiments showed a four-fold increase in Bodipy fluorescence after extraction of VSV G protein from the virus envelope with Triton X-100 or beta-octylglucoside. Bodipy-labelled virus retained its capacity to mediate fusion of viral membrane with phosphatidylserine liposomes. Incubation of Bodipy-VSV with proteases in the presence of detergent promoted a total fluorescence enhancement of ca. 20 fold, showing that the conjugate fluorescence was also sensitive to proteolysis. Fluorescence microscopy and flow cytometry experiments with macrophages incubated with Bodipy-VSV revealed that intracellular relaxation of fluorescence self-quenching resulted from a combination of viral disassembly due to pH-induced membrane fusion and viral protein degradation inside the endosomes. When macrophages were incubated simultaneously with ammonium chloride and protease inhibitors, the increase in fluorescence was abolished completely due to inhibition of both endosomal acidification and proteolysis. In addition, experiments carried out in the presence of protease inhibitors alone allowed, for the first time, isolated observation of G protein-mediated fusion of viral envelope with the endosomal membrane in living cells. The results indicate that this methodology may find wide application for further studies of viral infection.

Boron Compounds↗

PubMed1997

Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway.

The different partially folded states of the capsid protein that appear in the disassembly pathway of cowpea severe mosaic virus (CPSMV) were investigated by examining the effects of hydrostatic pressure, sub-zero temperatures and urea. The conformational states of the coat protein were analyzed by their intrinsic fluorescence, binding of bis(8-anilinonaphthalene-1-sulfonate) (bis-ANS) and susceptibility to trypsin digestion. CPSMV could be disassembled by pressure at 2.5 kbar. Intrinsic fluorescence and hydrodynamic measurements showed that pressure-induced dissociation was completely reversible. Virus pressurization in the presence of ribonuclease revealed that viral RNA was not exposed, since it was not digested by the enzyme, suggesting the maintenance of protein-nucleic acid interactions under pressure. When the temperature was decreased to -10 degrees C under pressure, CPSMV disassembly became an irreversible process and in this condition, viral RNA was completely digested by ribonuclease. These results suggest a relationship between protein-RNA interactions and CPSMV assembly. Bis-ANS binding and trypsin digestion of coat proteins revealed that they assume a different conformation when they are denatured by low temperatures under pressure or than when they are denatured by urea at atmospheric pressure. The results indicate that the coat proteins can exist in at least four states: (1) The native conformation in the virus capsid; (2) bound to RNA when the virus is dissociated by pressure at room temperature, assuming a conformation that retains the information for reassembly; (3) free subunits in a molten-globule conformation when the virus is dissociated by low temperature under pressure; and (4) free subunits completely unfolded by high concentrations of urea.

Anilino Naphthalenesulfonates↗

PubMed1996

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Animals↗

PubMed1996

Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding.

A theoretical model is presented that accounts for the facilitation of the pressure dissociation of R17 phage, and for the partial restoration of the concentration dependence of the dissociation, by the presence of subdenaturing concentrations of urea. As an indifferent osmolyte urea should promote the stability of the protein aggregates under pressure, and the decrease in pressure stability with urea concentration demonstrates that such indirect solvent effects are not significant for this case, and that the progressive destabilization is the result of direct protein-urea interactions. By acting as a "homogenizer" of the properties of the phage particles, urea addition converts the pressure-induced deterministic dissociation of the phage into a limited stochastic equilibrium. The model establishes the origin of the uniform progression from the stochastic equilibrium of dimers, to the temperature-dependent and partially concentration-dependent association of tetramers, to the fully deterministic equilibrium observed in many multimers and in the virus capsids.

Bacteriophages↗

PubMed1996

The use of hydrostatic pressure as a tool to study viruses and other macromolecular assemblages.

Recent studies on the effect of pressure on macromolecular assemblages have provided new information on protein-protein and protein-nucleic acid interactions. New findings have recently emerged on the use of hydrostatic pressure to assess intermediate states in the assembly pathways of viruses, multimeric proteins and protein-nucleic acid complexes, addressing many questions of macromolecular recognition.

Hydrostatic Pressure↗

PubMed1994

Arc repressor will not denature under pressure in the absence of water.

Hydration forces are believed to play a determining role in protein folding. We have examined the contribution of water for the stability of the native dimer state of Arc repressor, a DNA-binding protein. Hydrostatic pressure was utilized to convert Arc repressor protein from a native state to a denatured, molten-globule state at decreasing concentrations of water. The volume change associated with Arc denaturation fell linearly with the increase in concentration of glycerol, whereas the free energy of the reaction increased. The pressure that promotes 50% denaturation (p1/2) increased in direct proportion to the concentration of glycerol or the decrease of water. Extrapolated to zero concentration of water, the data indicate that pressure denaturation would not occur without water. It is concluded that water plays a crucial role in decreasing the stability of a protein to a level that is compatible with its biological properties.

DNA-Binding Proteins↗

PubMed1994

Differences in pressure stability of the three components of cowpea mosaic virus: implications for virus assembly and disassembly.

A comparison of pressure stability of empty capsids and ribonucleoprotein particles of cowpea mosaic virus (CPMV) is presented. A combination of high pressure and subdenaturing concentrations of urea was utilized to promote dissociation and denaturation. We found that RNA plays an important role in stabilizing the particles as well as in conferring reversibility to the pressure-induced denaturation. Dissociation and denaturation of the top component (empty capsid) was observed at 2.5 kbar and in the presence of 2.5 M urea. The pressure-dissociated state of the capsid protein had the characteristics of a denatured conformation as suggested by fluorescence spectra, lifetime of tryptophans, and binding of bis-ANS. The properties of the dissociated capsid protein were more similar to those of a molten-globule conformation, different from the more drastically unfolded state obtained using high concentrations of urea. Whereas the fluorescence of bis-ANS increased for the pressure-dissociated protein (1.5 M urea and 2.5 kbar), it decreased for the virus denatured by 6.0 M urea. Middle and bottom components underwent less than 50% change in center of spectral mass at 2.5 kbar and 2.5 M urea. The particles containing RNA could be fully affected by pressures of 2.5 kbar--as measured by the spectral shift--only in the presence of 5.0 M urea. RNA-containing capsids denatured by pressure did not bind bis-ANS, suggesting that the capsid protein continues to be bound to the RNA after the protein-protein contacts are broken by pressure. Reassembly of the nucleoprotein particles was obtained after decompression, reinforcing the idea that proteins had not dissociated from RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsid↗

PubMed1993

Reversible pressure dissociation of R17 bacteriophage. The physical individuality of virus particles.

In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.

Capsid↗