Proteolytic Processing of the Astrovirus Capsid

Abstract

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35 S]methionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10 5 -fold increase) after trypsin but not chymotrypsin treatment. This trypsinenhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.Astroviruses were first identified in the stools of children with gastroenteritis and characterized by electron microscopy (EM) (18). The particles were approximately 28 nm in diameter, and some had a star-like morphology. Since the initial observation in humans, similar viruses have been identified in a variety of other mammalian species. Lee and Kurtz first demonstrated growth of the virus in tissue culture and showed that trypsin is necessary for ongoing astrovirus replication (16). The development of enzyme immunoassays (EIAs) (11,12) and reverse transcriptase PCR assays has allowed larger numbers of samples to be screened with more-sensitive tests (13,14,21). It has become apparent that astroviruses are an important cause of gastroenteritis in a variety of settings, including nosocomial infections, daycare outbreaks, and diarrhea in outpatients (5-10, 19, 20, 24, 26).The astrovirus genome consists of a single-stranded positivesense RNA of approximately 7,200 nucleotides with three open reading frames (ORFs). The first, ORF1a, encodes a serine 3C type of viral protease (27). The second, ORF1b, is separated from ORF1a by a frame shift and encodes a viral RNA polymerase (17). The third, ORF2, encodes an 87-kDa polypeptide which functions as the capsid precursor. During infection, a subgenomic RNA that encodes the capsid precursor is found in abundance in the cell cytoplas...

Crystal structure of the human astrovirus capsid spike

“…Previously published SDS-PAGE gels show that the amount of VP34 present in infectious particles was comparable to that of VP27 and VP25 (10,12), indicating that most of the VP27 and VP25, although covalently independent, remain associated with the particle in a disordered manner. The loss of the connection of VP25 to the capsid shell, however, may allow VP25/VP27 spikes to wobble around, thus resulting in orientation disorder and their disappearance from the EM reconstruction on quasi-twofold axes.…”

Molecular characterization of a novel astrovirus associated with disease in mink

“…All astrovirus genomes show a rather conserved frameshift slippery sequence between ORF1a and 1b Marczinke et al, 1994), ensuring that the latter is only translated as a fusion polyprotein together with ORF1a, with an efficiency of 25-28 % when compared to the translation of ORF1a alone (Lewis & Matsui, 1996). The products from all three ORFs are posttranslationally cleaved into mature proteins (Bass & Qiu, 2000;Geigenmüller et al, 2002a;Gibson et al, 1998;Kiang & Matsui, 2002;Willcocks et al, 1999). However, the above information is derived mainly from studies with human astroviruses and similar data about animal astroviruses are still scarce.…”

“…The products from all three ORFs are posttranslationally cleaved into mature proteins The GenBank accession number of the sequence reported in this paper is AY179509. (Bass & Qiu, 2000; Geigenmüller et al, 2002a;Gibson et al, 1998;Kiang & Matsui, 2002;Willcocks et al, 1999). However, the above information is derived mainly from studies with human astroviruses and similar data about animal astroviruses are still scarce.…”

Lineage diversification and recombination in type-4 human astroviruses

“…By sequence comparison of HAstVs, the capsid protein precursor can be divided into a highly conserved N-terminal domain (aa 1-424), a hypervariable domain (HVR) (aa 425-688) and a highly acidic C-terminal domain (Wang et al, 2001). The mature infectious virion contains three predominant protein species derived from the N-terminal domain and from the HVR after intra-and extra-cellular processing (Bass and Qiu, 2000;Sanchez-Fauquier et al, 1994). Mapping of neutralising monoclonal antibodies (Sanchez-Fauquier et al, 1994) and structural analysis have revealed that AstV capsid spike is formed by dimerisation of a polypeptide spanning the HVR and a putative binding receptor site with affinity for polysaccharide molecules has been predicted in the spike structure (Dong et al, 2011).…”