A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector

Sialic acid-containing compounds play a key role in the initial steps of the paramyxovirus life cycle. As enveloped viruses, their entry into the host cell consists of two main events: binding to the host cell and membrane fusion. Virus adsorption occurs at the surface of the host cell with the recognition of specific receptor molecules located at the cell membrane by specific viral attachment proteins. The viral attachment protein present in some paramyxoviruses (Respirovirus, Rubulavirus and Avulavirus) is the HN glycoprotein, which binds to cellular sialic acid-containing molecules and exhibits sialidase and fusion promotion activities. Gangliosides of the gangliotetraose series bearing the sialic acid N-acetylneuraminic (Neu5Ac) on the terminal galactose attached in alpha2-3 linkage, such as GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are the major receptors for Sendai virus. Much less is known about the receptors for other paramyxoviruses than for Sendai virus. Human parainfluenza viruses 1 and 3 preferentially recognize oligosaccharides containing N-acetyllactosaminoglycan branches with terminal Neu5Acalpha2-3Gal. In the case of Newcastle disease virus, has been reported the absence of a specific pattern of the gangliosides that interact with the virus. Additionally, several works have described the use of sialylated glycoproteins as paramyxovirus receptors. Accordingly, the design of specific sialic acid analogs to inhibit the sialidase and/or receptor binding activity of viral attachment proteins is an important antiviral strategy. In spite of all these data, the exact nature of paramyxovirus receptors, apart from their sialylated nature, and the mechanism(s) of viral attachment to the cell surface are poorly understood.

The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl b-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus-cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell-cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells. INTRODUCTIONNewcastle disease virus (NDV) is one of the causes of avian respiratory diseases and economic losses in the poultry industry. It is a member of the family Paramyxoviridae, which includes enveloped, negative-stranded RNA viruses that encode two transmembrane glycoproteins: an attachment protein, HN, and a fusion protein or F protein. Virus adsorption occurs at the surface of the host cell membrane. The viral attachment protein HN recognizes and binds to sialic acid-containing molecules such as glycoproteins and glycolipids (Ferreira et al., 2004) and has neuraminidase and fusion-promoting activities. Following binding, the F protein promotes fusion between the viral and cellular membranes and the viral genome is delivered into the cytoplasm.Enveloped viruses enter the cell through two main pathways: direct fusion between the viral envelope and the plasma membrane; and receptor-mediated endocytosis. In the case of paramyxoviruses, it has been established that the membrane fusion process takes place at the host plasma membrane in a pH-independent manner. Despite this, it has been shown previously that fusion of NDV with cultured cells is enhanced at acidic pH (San Roman et al., 1999), leading us to propose that NDV may also penetrate the cell via an endocytic pathway in a pH-dependent process.After binding of an enveloped virus to its cognate receptor at the cell surface, membrane fusion is responsible for delivery of the nucleocapsid into the cytoplasm. Receptor binding and low pH have been considered to be the two main triggering mechanisms responsible for conformational changes in viral envelope glycoproteins leading to fusion. Also, it has been shown that in the recently emerged paramyxoviruses Nipah virus and Hendra virus, activation of the F protein is accomplished by proteolysis after endocytic entr...

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic schemewhere k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.Keywords: horeseradish peroxidase; differential scanning calorimetry; intrinsic fluorescence; circular dichroism; irreversible denaturation.Horseradish peroxidase (HRP) belongs to the superfamily of the heme-containing plant peroxidases (EC 1.11.1.7), which has been divided into three classes [1], supported in the first instance by comparison of amino-acid sequence data and confirmed by more recent data on crystal structures [2]. Plant peroxidases, including HRP, comprise class III of the superfamily. Although the function of peroxidases is often seen primarily in terms of the conversion of H 2 O 2 to H 2 O, this should not be allowed to mask their wider participation in other reactions, many of which are biologically significant. Despite the enormous interest in peroxidases owing to their broad practical applications in biotechnology, the data concerning their structural stability are sparse. Although several publications have addressed the thermal stability of peroxidases [3±8], to date the mechanism of the process of thermal denaturation remains unclear. It is known that the biological functions of proteins depend on the correct folding of their native structure and that loss of this folded structure leads to an unfolded, inactive state. Consequently, the study of protein stability is important both from the academic and applied points of view.Factors affecting conformational stability have been studied most intensively in proteins under reversible conditions [9±15]. Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturat...

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M.

Thermal denaturation of Torpedo culifornicu acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the twostate kinetic scheme N 5 D, with activation energy 131 * 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 t 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.Keywords: acetylcholinesterase; differential scanning calorimetry; irreversible denaturation; molten globule; thioldisulfide exchange; two-state kinetic model Folding of many small globular proteins is a cooperative process in the course of which only two states, the fully unfolded state, U, and the native state, N, are significantly populated. In the case of large proteins, which are generally believed to contain several domains (Jaenicke, 1991;Garel, 1992), folding has generally been considered to be cooperative within each domain, the only species populated in the course of either folding or unfolding being combinations of completely folded and completely unfolded domains (Privalov, 1982;Brandts et al., 1989;Garel, 1992). Evidence is accumulating, however, that another state, intermediate between N and U, can exist. This is a compact state that lacks the unique tertiary structure of the native protein but possesses substantial secondary structure. This state has been named the molten globule (MG) state (Kuwajima, 1989;Kim & Baldwin, 1990;Ptitsyn, 1992). The MG state is considered to serve as an intermediate on the pathway from the nascent polypeptide chain to the fully folded native protein in vivo (Gething & Sambrook, 1992). For most proteins, the MG state is unstable under physiological conditions and readily converts to the N state in vitro (Ptitsyn, 1992 Because the definition of the MG state is controversial (see, for example, Griko et al., 1994; Ewbank et al., 1995;Okazaki et al., 1995), in the present paper this term is used to refer to compact states of acetylcholinesterase (AChE) that preserve substantial secondary structure and lack most of the tertiary structure of the native enzyme. We have shown recently that exposure of a native dimeric form of Torpedo AChE to various treatments generates long-lived partially unfolded species displaying many of the characteristics of the MG...

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.

The stability of a lentil lectin, an all-beta protein, has been perturbed by changes in pH and temperature. In the pH interval 5.0 --> 10.0, the overall secondary structure does not undergo significant changes. However, if the individual components of the infrared amide I band are considered, changes in band components attributed to variations in beta-sheet and beta-turns cross-interactions are detected. The combined effects of pH and temperature reveal that the protein is more compact at pH 7.5 with lower denaturation temperatures at pH 5.0 or 10.0, indicating a less stable protein under those conditions. According to our results, the structural stability of the beta-sheet would depend not only on the intermolecular interactions among the strands but also on the conformation of the segments connecting these strands. The protein infrared band assignment has also been examined since the three-dimensional structure of the lentil lectin protein is known from X-ray diffraction studies. Two of the bands observed are attributed to beta-sheet. The one at 1620 cm-1, not affected if the medium is deuterated, is assigned to hairpins composed by two strands connected by a rigid turn whereas that located at 1633 cm-1 corresponds to strands associated by more flexible segments. The band appearing at 1645 cm-1 in H2O corresponds to the open, flexible loops that are connecting the beta-strands. The simplest assumption of the various secondary structure components having identical IR extinction coefficients is enough to provide IR-derived data that are in good agreement with the structure solved by X-ray diffraction.