Molecular biology of astroviruses selected highlights

Suzanne M. Matsui, David Kiang, Nancy Ginzton, Teri Chew and Ute Geigenmuller-Gnirke

Department of Medicine, Division of Gastroenterology, Stanford University School of Medicine, Stanford, CA 94305-5487, and Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA

Abstract. Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5—6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates that viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length cDNA clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORFla. These studies demonstrate that the full-length ORFla product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

2001 Gastroenteritis viruses. Wiley, Chichester (Novartis Foundation Symposium 238) p219-236

Human astrovirus is the prototype of the Astroviridae, a family of non-enveloped, positive-strand RNA viruses (reviewed in Matsui & Greenberg 2000). By direct electron microscopy, astroviruses recovered from stools display a distinctive surface star for which they were named. Since the star-like appearance is evident on approximately 10% of the viral particles of a given preparation, an experienced microscopist may be required to make a definitive identification on the basis of morphology alone. In addition, in a study of astroviruses propagated in cell culture, the surface star was not found, but could be induced by alkaline treatment (Risco et al 1995). In this study, electron micrographs of intact purified viral preparations (not alkaline-treated) showed particles that were round with spike-like protrusions from the surface and an external diameter of 41 nm.

FIG. 1. Human astrovirus serotype 1 map from negative stain (Yeager et al 2001). The result of image processing (Yeager et al 1990) of a negatively stained preparation of purified H-Ast1 virions is shown. Image processing requires digitization of micrographs, masking of individual particles, subtraction of background densities, and calculation of the Fourier transforms for each particle. Level of resolution is 24 A. A particle, viewed along the fivefold axis of symmetry, is shown.

FIG. 1. Human astrovirus serotype 1 map from negative stain (Yeager et al 2001). The result of image processing (Yeager et al 1990) of a negatively stained preparation of purified H-Ast1 virions is shown. Image processing requires digitization of micrographs, masking of individual particles, subtraction of background densities, and calculation of the Fourier transforms for each particle. Level of resolution is 24 A. A particle, viewed along the fivefold axis of symmetry, is shown.

Recent structural analysis using cryoelectron microscopy shows some unique features of the astrovirus surface that cannot be appreciated in most routine images obtained by direct electron microscopy (Yeager et al 2001). Images of frozen-hydrated astrovirus particles, purified from a preparation of cell culture-adapted human astrovirus serotype 1 and stained with uranyl acetate, show spherical particles of uniform size with clearly visible surface spikes. 3D reconstruction from these cryoelectron microscopy images show a smoothly rippled, solid capsid shell with a diameter of 330 A and 30 dimeric spikes, centred at the twofold axis of symmetry, that extend about 50 A from the surface (Fig. 1). Inside the capsid, the genomic RNA appears to assume a partial icosahedral configuration.

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