Association of the astrovirus structural protein VP90 with membranes plays a role in virus morphogenesis

Abstract

VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (mVP90), as well as in fractions enriched in cytosolic proteins (cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both mVP90 and cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with mVP90, but not with cVP90, suggesting that mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.

FIG. 1.

Structural astrovirus proteins VP90 and VP70 are differentially located in the cell. Cytoplasmic extracts were fractionated by ultracentrifugation in density gradients, as described in Materials and Methods. Twelve fractions were collected and analyzed for immunoblotting with anti-Yuc8 antibodies in a 7.5% polyacrylamide gel (A) and for the presence of infectious particles after trypsin treatment (B). Viral proteins are marked on the right.

FIG. 2.

Protein VP90 is found soluble and associated with membranes in the cells. Cytoplasmic extracts of untreated and infected cells (A) or cells treated for 30 min with TX-100 at room temperature (B) were fractionated by density gradients, and fractions were separated by 7.5% SDS-PAGE and immunoblotted with anti-TYVD antibodies. In vitro-translated VP90 labeled with ³⁵S-Express label in the absence (C) or in the presence (D) of microsomes or in the presence of microsomes but with previous treatment with TX-100 (E) was loaded in the density gradients. ORF1a was in vitro translated in the presence of microsomes, and the p20 amino-terminal product of nsp1a was immunoprecipitated with anti-1a-1 antibodies (F) (16). Viral proteins in panels C to F were separated by SDS-PAGE and detected by autoradiography. The viral proteins VP90, VP70, and p20 are marked at the right.

FIG. 3.

The carboxy terminus of VP90 is involved in membrane association. Cytoplasmic extracts treated with trypsin (200 μg/ml) for 30 min at 37°C were fractionated by density gradients and immunoblotted with the indicated antibodies. Viral proteins are marked at the right and molecular mass markers (in kilodaltons) at the left.

FIG. 4.

Membrane-associated VP90 is less susceptible to trypsin digestion. Density gradients of untreated (A) or Z-VAD-FMK-treated (B) infected cells were obtained as described for Fig. 2, and fractions were immunoblotted with anti-TYVD antibodies. Fractions 2 and 9, corresponding to the cytosolic (cyt) and membrane-associated (memb) fractions from these gradients, were treated with the trypsin concentrations and immunoblotted with anti-E4 antibodies, as indicated (C and D). Digestion mixtures were separated in 12.5% (C) and 7.5% (D) polyacrylamide gels to observe products in a wide molecular mass range. Trypsin products of ^cVP90 from Z-VAD-FMK-treated cells were immunoblotted also with anti-Yuc8 antibodies to ensure that the protein was not totally degraded by trypsin digestion (E). In every panel, viral proteins are marked at the right. A putative oligomer of VP90 is also marked. Numbers at left in panels A to D are molecular masses in kilodaltons.

FIG. 5.

Astrovirus capsid proteins form oligomers. VP90s of HAstV serotypes 1 and 8 were translated in vitro in the presence of [³⁵S]Met and electrophoresed in 7.5% polyacrylamide gels (A). Samples were boiled or not boiled, as indicated, in the presence of reducing agents and analyzed by autoradiography. Two fractions of cesium chloride gradients from HAstV-8-infected-cell lysates, obtained in the absence of trypsin and the presence of fetal bovine serum, were separated by PAGE and analyzed by immunoblotting with anti-E2 antibodies (B). Purified particles were from fractions of densities of around 1.30 (lane 1) or 1.36 (lane 2) g/cm³. The molecular weight markers (weights in thousands) and the viral proteins are marked.

FIG. 6.

Electron micrographs of HAstV-8-infected Caco-2 cells. Cells were harvested at 24 hpi and processed for electron microscopy, as described in Materials and Methods. Panels A, C, and E represent three different cells, and panels B, D, and F represent enlargements of the corresponding areas. Astrovirus particles were observed in clusters (VP) and in isolated (circled) forms. Particles that look partially assembled inside or at the edges of vesicles (V) induced during infection are marked with arrows. Bars, 200 nm.

FIG. 7.

IEM of Caco-2 cells infected with HAstV-8. Cells were infected with the Yuc8 astrovirus strain at a multiplicity of infection of 3 for 24 h, fixed, and processed for IEM with the indicated primary antibodies. Goat anti-rabbit IgG labeled with 10-nm gold particles was used as detection antibody. Cells in panels A to D were processed with anti-Yuc8, and cells in panels E and F were processed with anti-E4 antibodies. The photograph shown in panel E is from Z-VAD-FMK-treated cells. V and VP are used for vesicles and viral particles in clusters, respectively. Arrows indicate positive gold signals. Bars, 200 nm.

FIG. 8.

IEM of membrane-enriched fraction. Fraction 9 of iodixanol gradients was processed for IEM using anti-E4 antibodies and goat anti-rabbit IgG labeled with 10-nm gold particles, as described in Materials and Methods. Images of different preparations of vesicles with particles associated are shown. Arrows indicate positive immunogold signals. Images of vesicles containing associated particles in the upper panels (white rectangles) are enlarged in the lower corresponding panels.

FIG. 9.

Nonstructural proteins and viral RNA colocalize with ^mVP90. (A and B) Fractions of the iodixanol gradients were immunoblotted with antibodies to the recombinant proteins 1a-3 (A) and 1b-2 (B), as indicated. Viral proteins and molecular weight markers (weights are in thousands) are marked. (C) IEM of Caco-2 cells using anti-1a3 as the primary antibody, as mentioned in Materials and Methods. V and VP are used for vesicles and viral particles, respectively. Arrows indicate positive signals. Bars, 200 nm. (D) Viral RNA was obtained from iodixanol gradient fractions 2 (cyt; lanes 1, 2, 6, and 7) and 9 (memb; lanes 3, 4, 8, and 9) of mock-infected (lanes 1, 3, 6, and 8) and HAstV-infected (lanes 2, 4, 7, and 9) cells or from supernatant of infected cells harvested 24 hpi (lanes 5 and 10). RT was carried out with oligonucleotide Mon244 to detect the negative-sense RNA (lanes 1 to 5, 11, 12, and 15) or Mon245 to detect the positive-sense RNA (lanes 6 to 10, 13, 14, and 16). As controls, Superscript was heated at 85°C for 10 min before the RT reaction (lanes 12 and 14) and no reverse transcriptase (RTase) was added to the RT-PCR (lanes 15 and 16). The molecular weight marker is φX174 digested with HaeIII.

FIG. 10.

Model of the astrovirus morphogenesis pathway. (A) Scheme of VP90, in which the conserved (white box), the hypervariable (horizontally hatched), and the acidic-rich (vertically hatched) regions are indicated. Downward arrowheads and upward arrows indicate trypsin and caspase cleavages, respectively. The regions of VP90 comprised in the constructs employed to generate the antibodies used in this work are shown below the scheme of VP90. (B) Model for the astrovirus morphogenesis process (see text).