SUMMARYMass determinations on highly purified influenza virus preparations were performed using the technique of scanning transmission electron microscopy. The masses of the three strains, X49, B/Singapore/222/79 and B/Hong Kong/8/73 were determined. The average value was 174 × 106 daltons with only small differences between the three strains. The mass of virus particles after removal of the protruding spike proteins, haemagglutinin and neuraminidase by bromelain treatment was determined to be 86 × 106 daltons. From the mass difference and the known molecular weight of the spike proteins the number of spikes was estimated to lie in the range 400 to 500.Influenza virus is a complex virus belonging to the Orthomyxoviridae. The virus is composed of protein, lipid, carbohydrate and RNA; there are four major proteins involved in the virus architecture. The outer virus envelope consists of a lipid bilayer from which the so-called spikes project externally. These spikes consist of trimers and tetramers of haemagglutinin (HA) and neuraminidase (NA) respectively. The inner core of the virus consists of M protein, nucleoprotein and RNA. The molecular biology of the virus was reviewed recently (Webster et al., 1982).There is no general agreement on the size, shape and exact composition of the virus. Most of the information on its structure was obtained from biochemical studies (Schulze, 1973;Laver, 1973), electron microscopic results (Wrigley, 1979;Nermut & Frank, 1971), X-ray crystallographic work (Wilson et al., 1981 ;Varghese et al., 1983) and solution scattering data (Mellema et al., 1981). Here we describe the determination of particle weight and spike content by scanning transmission electron microscopy (STEM). This method consists of scanning a focused electron beam across the specimen and obtaining a digital signal from the scattered electrons that can be computer-processed. Under certain well defined conditions the scattering intensity is proportional to the scattering mass (see Freeman & Leonard, 1981).As proposed by Freeman & Leonard, we determined particle masses by using an internal standard. Influenza particle masses were determined by comparison with tobacco mosaic virus which has a very well known mass per unit length. A linear relationship was assumed between the scattering mass and the scattering intensity; this assumption holds under our specific measurement conditions (acceleration voltage, carbon film thickness, etc.) up to a thickness of approximately 60 nm. From shadowing experiments the thickness of the unstained, flattened, influenza particles was found to be 48 + 4 nm, which is in the linear range. The validity of the mass determination method was indicated by the very good reproduction of published data on masses of a number of plant viruses with well known molecular weights. A full description of our implementation of the STEM mass determination method will be published elsewhere (R. W. H. Ruigrok et al., unpublished results).Influenza virus preparations were obtained from DUPHAR (Weesp, The Net...

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A model is postulated for the alfalfa mosaic virus (AlMV) coat protein ( M , 24250), consisting of a rigid core and a flexible N-terminal part of approximately 36 amino acid residues. This model is based on a proton nuclear magnetic resonance study of the AlMV coat protein. The NMR spectrum of the coat protein shows several motionally narrowed resonances which strongly decrease in intensity when 25 N-terminal amino acid residues are removed by a mild tryptic treatment. The motionally narrowed resonances, therefore, are assigned to the N-terminal region. Specific assignments in the spectrum for tyrosine-21, threonine-15 and the N-terminal N-acetyl group can be made. The model is supported by photochemically induced dynamic nuclear polarization experiments on the intact protein and the trypsinated protein, which indicate that tyrosine-21 is the only tyrosine which is easily accessible from the solvent. Furthermore, NMR spectra were obtained from a polymerisation product of the coat protein, the so-called 12-S product in which only the sharp resonances of the N-terminal part are observed. Apparently, due to the large particle mass (about 400 kDa), the resonances of the core part of the protein are broadened to such an extent that they are no longer observable. The part of the spectrum which originates from the mobile region of the coat protein can be simulated by calculating a spectrum for a random coil peptide of about 36 N-terminal amino acids. This indicates that the flexible region extends to about 36 amino acid residues from the N terminus and that it probably occurs in a random coil structure. Evidence is presented for a change in structure of the protein in solution when it assembles into the virus particle. This conclusion is based on an observed spectral change of histidine-220.Alfalfa mosaic virus (AlMV) is a plant virus with a tripartite single-stranded RNA genome, encapsidated by one type of coat protein into cylindrical particles of different length with rounded ends. A number of aspects of the structure and assembly of this virus have been described in earlier publications [1,2]. The amino acid sequence of the coat protein is known [3]. Besides its structural function, the coat protein ( M , 24250) plays an essential role in the infection process [4].To obtain a better understanding of this dual nature of the coat protein, knowledge of its structure will be of vital interest. The coat protein is capable of polymerizing into a so-called 3 0 3 particle, a regular spherical ( T = 1) particle, which was shown to consist of 60 copies of the coat protein [5]. Recently, the crystallisation of 30-S particles after mild tryptic digestion has been reported [6] and a X-ray diffraction study is being carried out. However, the tryptic treatment, by which 25 N-terminal amino acid residues are removed, results in a protein that is no longer biologically active [7]. Furthermore, X-ray crystallographic studies do not give information about those parts of the molecule that are in static or dynamic disorder [8] and add...

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