Processing of Nonstructural Protein 1a of Human Astrovirus

Abstract

Astrovirus contains three open reading frames (ORF) on its genomic RNA, ORF1a, ORF1b, and ORF2. ORF1a encodes a 920-amino-acid (aa) nonstructural protein, nsP1a, which displays a 3C-like serine protease motif. Little is known about the processing of nsP1a or whether the protease it contains is active and involved in autocatalytic processing. Here we address both of these matters. Intact and N-terminally deleted forms of ORF1a from human astrovirus serotype 1 were expressed in BHK cells, and nsP1a-derived processing products were immunoprecipitated with an nsP1a-specific antibody or an antibody specific for an N-terminally linked epitope tag. The mapping of the main processing products, p20 and p27, suggests cleavage sites near aa 170, 410, and 655 of nsP1a. Cleavages at around aa 410 and 655, but not aa 170, were abolished when a 9-aa substitution was introduced into the protease motif in nsP1a. The p27 processing product was also found in Caco-2 cells that had been infected with human astrovirus serotype 1, confirming the presence of the cleavage sites at approximately aa 410 and 655.

Protein Products of the Open Reading Frames Encoding Nonstructural Proteins of Human Astrovirus Serotype 8

“…The full-length polyproteins nsp1a and nsp1ab, with calculated molecular masses of 103 and 160 kDa, respectively, were not found in cell lysates from Yuc8-infected or plasmid-transfected cells, suggesting that cotranslational processing occurs during their synthesis, as previously shown for HAstV-1 in transient expression experiments (5). Two proteins of around 19 to 20 kDa seem to represent the N-terminal products of the cotranslational processing of polyproteins nsp1a and nsp1b during astrovirus infection.…”

“…Few studies have examined the synthesis and processing of the HAstV nonstructural proteins, and many details of these events remain to be elucidated (5,7,11,24). In this study we contribute to the characterization of the processing of the nonstructural polyproteins of astroviruses, using as a model an HAstV-8 strain.…”

“…Although studies of the processing of the nonstructural polyproteins of HAstV have been carried out in several laboratories, this event is still poorly understood (5,7,11,24). The identification of the final and intermediate proteins which result from the proteolytic cleavage of the full-length nonstructural protein precursors has been hampered by the lack of shutoff of the cellular protein synthesis by the virus (19) and because antisera to specific fragments of the nonstructural region have not been available.…”

“…The processing of the astrovirus nonstructural polyproteins has not been completely characterized, and some of the reported data are conflicting (5,7,11,24). Willcocks et al (24) detected products of 75, 34, 20, 6.5, and 5.5 kDa in HAstV-1-infected cells by using antibodies to the carboxy-terminal end of nsp1a (amino acid residues 643 to 940), suggesting that the primary translation product of ORF1a is processed from its amino terminus, and also detected a protein of 59 kDa by using antibodies to nsp1b.…”

“…Willcocks et al (24) detected products of 75, 34, 20, 6.5, and 5.5 kDa in HAstV-1-infected cells by using antibodies to the carboxy-terminal end of nsp1a (amino acid residues 643 to 940), suggesting that the primary translation product of ORF1a is processed from its amino terminus, and also detected a protein of 59 kDa by using antibodies to nsp1b. Geigenmuller et al (5) reported proteins of approximately 20 and 27 kDa as final products of nsp1a by transient expression of HAstV-1 cDNA clones and demonstrated that the cleavages at around amino acid residues 410 and 655 of nsp1a, which generate the 27-kDa protein, were dependent on the viral serine protease. Using an in vitro translation assay for HAstV-2 nsp1a and nsp1ab proteins, Gibson et al (7) could not identify any processed products and suggested that the viral serine protease could require a cellular factor, not present in the reticulocyte lysate, for its activity.…”