Proteolytic processing of a serotype 8 human astrovirus ORF2 polyprotein

Abstract

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.

FIG. 1.

Sera to recombinant Yuc8 proteins recognize 90- and 70-kDa proteins in Yuc8-infected cells. A monolayer of Yuc8-infected Caco-2 cells was washed with PBS twice at 24, 48, and 120 h postinfection (hpi) and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% SDS, plus phenylmethylsulfonyl fluoride and leupeptin as protease inhibitors. A lysate of mock-infected cells was included as a control. Cell lysates were homogenized and centrifuged at 14,000 × g to discard the pellet. Supernatant was collected and loaded in an SDS-15% polyacrylamide gel for immunoblot analysis with the indicated sera. The migration of the molecular mass markers (in kilodaltons) (indicated by a protein ladder [GIBCO] for anti-E1 and prestained protein standards [GIBCO] for anti-E2 and anti-E3) and of the viral proteins is indicated.

FIG. 2.

Trypsin treatment of purified Yuc8 particles cleaves VP70 into several polypeptides and enhances the virus infectivity. Purified Yuc8 astrovirus was treated with the indicated concentrations of TPCK-treated trypsin for 1 h at room temperature. Each sample was then divided in two portions which were either run in an SDS-12.5% polyacrylamide gel and stained with silver (A) or used to determine viral infectivity (B). (A) Trypsin alone (400 μg/ml) was used as a control, migrating as a 25-kDa protein (rightmost lane). The molecular mass standards (in kilodaltons) (protein ladder; GIBCO) and the positions of the viral proteins are marked. The assignment of the molecular mass for the viral proteins was made based on their average migration in several experiments and different standards, as reference. The infectivity was determined by quantification of the infected cells detected by immunofluorescence and expressed as n-fold enhancement.

FIG. 3.

Processing of the VP70 protein by trypsin is ordered. Purified Yuc8 astrovirus particles were treated with the indicated trypsin concentrations, as mentioned in Fig. 2, and each sample was divided in equal parts to run three independent SDS-12.5% polyacrylamide gels and transferred to nitrocellulose. Each blot was incubated with either anti-KSN (A), anti-E2 (B), or anti-E3 (C) antibodies. As a control, 100 μg of trypsin/ml was included (rightmost lane in panels B and C). Note that anti-E2 (B), but not anti-E3 (C), partially cross-reacts with trypsin (25 kDa). An additional ∼69-kDa band detected by anti-E3 (C) is not of viral origin, since it appears in the trypsin-alone lane. The position of the viral proteins and of trypsin are indicated as well as of the molecular mass markers (protein ladder; GIBCO). The infectivities of these samples were also determined, confirming that the enhancement of infectivity increased slightly with trypsin at 10 μg/ml and was higher at 100 μg/ml, as shown in Fig. 2.

FIG. 4.

N-terminal sequence of VP28-derived proteins. Purified Yuc8 particles were trypsin treated and electroblotted to PVDF membranes. VP28, VP27, and VP25 were purified, and the N termini were sequenced. The sequence alignment of eight HAstV serotypes between residues 287 and 459 of ORF2 is shown. The cleavage sites for the proteins VP28, VP27, and VP25 (boxes marked by black arrows) and the amino acid residues which could potentially represent the carboxy end of the VP41-derived proteins (boxes at the amino acid residues 299, 313, 347, 354, 359, 361, and 365) are indicated. The cleavage sites for the proteins VP29 and VP26 of HAstV-2, previously described (20), are also indicated (white arrows). The dots along the sequence denote identity, and only amino acid changes are marked. The previously described variable regions VR1 and VR2 and part of VR3 (16) are underlined. The common astrovirus epitope predicted (21) is marked with asterisks. The numbers below the sequences indicate the amino acid position based on the Yuc8 sequence. Sequence alignment was made by Clustalw analysis (http://www.ebi.ac.uk/clustalw/) using sequences with accession numbers L23513 (HAstV-1), A45695 (HAstV-2), AF141381(HAstV-3), Z33883 (HAstV-4), U15136 (HAstV-5), Z46658 (HAstV-6), Z66541 (HAstV-8), and AF260508 (Yuc8).

FIG. 5.

(A) Diagram of the ORF2 of HAstV and recombinant astrovirus proteins. ORF2 is represented as a box, and the recombinant proteins and the KSN peptide are represented as thick black lines. The diagram is to scale, and the relative positions of the astrovirus Yuc8 recombinant proteins E1, E2, and E3 and the peptide KSN is shown (subscripts indicate the amino acid residues included in each protein). The hypervariable regions found among human astrovirus serotypes (16) are shown as striped boxes. The arginine (R) residues identified by Bass (2) and Sanchez-Fauquier (20) as cleavage sites in the capsid polyprotein of HAstV-1 and HAstV-2 and the protein products proposed (in parentheses) to be generated by these cleavages are indicated. Asterisks represent susceptible trypsin sites conserved among astroviruses belonging to different serotypes, which could be cleaved to yield the VP41- and VP28-derived polypeptides. (B) Proposed trypsin processing pathway for the ORF2 polyprotein of astrovirus Yuc8. Boxes represent the products observed during virus activation; the final products, present in fully activated particles, are indicated in boldface type. The N-terminal amino acid residue of the VP28-derived products is shown. See details in the text.