Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.Human astroviruses (HAstV) have been found to be a frequent cause of gastroenteritis among young children worldwide (5,7,8,15). The virions are formed by a nonenveloped protein capsid and a positive-stranded RNA genome of approximately 7 kb (9, 22). The RNA genome has three open reading frames (ORFs) (ORF1a, -1b, and -2), each encoding at least one polyprotein. The ORF1a contains viral serine protease and nuclear localization signal motifs, whereas the ORF1b has an RNA-dependent RNA polymerase motif (9,22). The products of ORF1a and ORF1b are synthesized from the genomic RNA as two polyproteins, with the latter being produced as a polyprotein 1a-1b (approximately 160 kDa) through a frameshift translational mechanism (12)(13)(14). It is believed that the products of these ORFs, processed to smaller polypeptides by the viral protease, are involved in the viral RNA replication. ORF2, of approximately 780 amino acid residues, depending on the strain (21), codes for the structural virus polypeptides (18). The structural polyprotein is translated from a polyadenylated subgenomic RNA produced at high levels during infection, which is 3Ј-colinear with the genomic RNA (17). Based on the homology among strains belonging to different serotypes, at least two domains in the product of this ORF have been predicted (16,21). The first domain includes amino acid residues 1 to 415, and it is highly conserved among all the human serotypes and some viruses from animal origin; the second domain (amino acid 416 to the end) is highly divergent among human serotypes (10,16,21,23). Neutralizing epitopes have been mapped to the second domain (3, 20); therefore, it is likely that this hypervariable region is exposed on the viral particle. It is known that astrovirus in...