ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 341, No. 1, May 1, pp. 89–97, 1997 Article No. BB979953 A Differential Scanning Calorimetric Study of Newcastle Disease Virus: Identification of Proteins Involved in Thermal Transitions 1 Valery L. Shnyrov, 2,3 Galina G. Zhadan, 2 Ce ´sar Cobaleda, Ana Sagrera, Isabel Mun ˜ oz-Barroso, and Enrique Villar 3 Departamento de Bioquı B mica y Biologı B a Molecular, Universidad de Salamanca, 37007 Salamanca, Spain Received November 26, 1996, and in revised form February 12, 1997 Newcastle disease virus (NDV), 4 a member of the The irreversible thermal denaturation of Newcastle family of Paramixoviridae, is composed of an outer lipo- disease virus was investigated using different tech- protein envelope and an internal helical nucleocapsid niques including high-sensitivity differential scanning (1). The nucleocapsid core contains a single-strand neg- calorimetry, thermal gel analysis intrinsic fluores- ative RNA, the NP nucleoprotein (55 kDa), the P phos- cence, and neuraminidase activity assays. Application phoprotein (53 kDa), and the L polymerase protein (200 of a successive annealing procedure to the scanning kDa). The envelope is composed of a lipid bilayer, which calorimetric endotherm of Newcastle disease virus is acquired from the host plasma membrane, and has furnished four elementary thermal transitions below two transmembrane glycoproteins: the F protein (55 the overall endotherm; these were further identified kDa), which promotes virus–cell fusion, and the HN as coming from the denaturation of each viral protein. protein (74 kDa), having hemagglutinating and neur- The shape of these transitions, as well as their scan- aminidase activities (2, 3). The possible presence of a rate dependence, was explained by assuming that specific site on the HN protein that interacts with the thermal denaturation takes place according to the ki- F protein in the fusion process has also been demon- netic scheme N r k D, where k is a first-order kinetic strated (4, 5). This type of virus can penetrate cells constant that changes with temperature, as given by through direct fusion with the plasma membrane at the Arrhenius equation; N is the native state; and D is neutral pH values (6, 7). There is another protein, im- the denatured state. On the basis of this model, activa- mediately below the viral envelope: the inner, nongly- tion energy values were calculated. The data obtained cosylated M matrix protein (40 kDa), which interacts with the other methods used in this work support the electrostatically with the cytoplasmic part of the HN proposed two-state kinetic model.  1997 Academic Press protein and with the contents of the nucleocapsid (8, 9), Key Words: calorimetry; denaturation; thermal gel thus conferring rigidity to the virion structure. There is analysis; Newcastle disease virus. no detailed information about the relative concentra- tions of individual proteins in intact NDV. It is only known that the M, F, and HN proteins are of equimolar concentration and that, together, they represent ca. 1 This study was supported by Grant PB 93-0629 from the Spanish 90% of the total viral protein (10). DGICYT to E.V.; G.G.Zh. is a sabbatical leave recipient from the Paramyxoviruses have frequently been character- Spanish DGICYT (Ref. SAB95-0118); C.C. and A.S. are fellowship holders of the Plan de Formacio ´n del Profesorado Universitario y ized in terms of the thermal stability of their hemagglu- Personal Investigador from the Ministerio de Educacion y Ciencia, tinating activity (HA), their neuraminidase activity Spain, and the Universidad de Salamanca, Spain, respectively. (NA), and their infectivity (11, 12). It has been shown 2 Permanent address: Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia. 4 Abbreviations used: NDV, Newcastle disease virus; HA, hemag- 3 To whom correspondence should be addressed at the De- partamento de Bioquı B mica y Biologı B a Molecular, Universidad de Sa- glutinating activity; NA, neuraminidase activity; DSC, differential scanning calorimetry; BSA, bovine serum albumin; TGA, thermal lamanca, Avenida del Campo Charro, s/n, 37007 Salamanca, Spain. Fax: /34-23-294579. gel analysis. 89 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.