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 [35S]Smethionine, 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 trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.

FIG. 1

Pulse-chase analysis of processing of the serotype 1 astrovirus capsid in the absence of trypsin. Caco-2 cells in 10% FBS were infected for 12 h with astrovirus, pulsed for 30 min with [³⁵S]methionine, and then chased with 10× methionine. Supernatants and cell lysates were harvested at 0, 2, and 4 h after completion of the pulse and immunoprecipitated with MAb 5B7. The supernatant portion was exposed to film four times longer than the cell lysates were. Molecular size markers (in kilodaltons) are indicated.

FIG. 2

Human serotype 1 astrovirus particles grown in the presence (A) or absence (B) of trypsin with negative staining with phosphotungstic acid. Particles were grown and purified as described in Materials and Methods.

FIG. 3

Protein composition of serotype 1 astrovirus particles by SDS-PAGE. Virus was purified as described in Materials and Methods. Lanes: IP, viral protein immunoprecipitated with MAb 5B7 from trypsin-free astrovirus infection; NT, virus grown in the absence of trypsin; T, virus grown in the presence of trypsin at 10 μg/ml; NT+T, virus grown in the absence of trypsin and subsequently treated for 30 min at 37°C with trypsin at 10 μg/ml. Estimated molecular masses of astrovirus proteins (left) are indicated, as well as molecular size markers, in kilodaltons.

FIG. 4

Enhancement of astrovirus infectivity by trypsin or chymotrypsin (chymo) treatment. Purified, trypsin-free astrovirus was incubated with the indicated concentrations of trypsin or chymotrypsin (Nα-p-tosyl-l-lysine chloromethyl ketone [TLCK] treated; Sigma-Aldrich, St. Louis, Mo.) at 37°C for 30 min prior to titration on Caco-2 cells by immunoperoxidase focus assay. The experiment was performed three times with three replicate titrations each time. Shown are mean values from a typical experiment. Error bars indicate 1 standard deviation.

FIG. 5

Reactivity of various antibodies with trypsin-treated (ASTRO +T) and non-trypsin-treated (ASTRO −T) astrovirus 1. Plates were precoated with trypsin-treated or trypsin-free astrovirus or rhesus rotavirus (ROTA) and then probed with various antibodies as described in Materials and Methods. Bars indicate the mean A₄₅₀ of four replicate wells for each condition. Error bars indicate 1 standard deviation.

FIG. 6

Schematic model of proposed astrovirus capsid processing. Astrovirus ORF2 is translated as an 87-kDa protein. Within the cell, the 70 amino-terminal amino acid residues are removed, probably by a cellular protease. This may facilitate capsid formation. Outside the cell, trypsin cleavage at sites which include residue 395 results in the mature, infectious virion consisting of three proteins of approximately 26, 29, and 34 kDa.