The formation of viroplasm-like structures by the rotavirus NSP5 protein is calcium regulated and directed by a C-terminal helical domain

Abstract

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.

FIG. 1.

Truncation of the C-terminal 30 residues from NSP5 abolishes its insolubility and hyperphosphorylation. (A) N-NSP5 lacking the C-terminal 30 residues (ΔC-30) was expressed in COS-7 cells, and soluble and insoluble fractions were collected 48 h posttransfection. Protein expression in each fraction was analyzed by Western blotting with anti-HisG antibody. Unlike full-length N-NSP5 (N-NSP5 wt) (lanes 1 and 3), the ΔC-30 mutant was detected only in the soluble fraction (lanes 2 and 4). (B) Cells expressing either full-length N-NSP5 or the ΔC-30 mutant were analyzed as described above after treatment with the phosphatase inhibitor calyculin A (+) or with no treatment (−). The brackets denote the basal (b) and hyperphosphorylated (hp) isoforms of NSP5. Numbers refer to the migration of protein markers, and the asterisk indicates a nonspecific cellular protein.

FIG. 2.

Rotavirus NSP5 protein contains a conserved predicted amphipathic α-helix within the carboxyl 30 amino acids. (A) Prediction of the secondary structure present in the carboxyl 37 amino acids of the RRV NSP5 protein (AA) using the profile network Heidelberg (PROF_sec) method. Numbers indicate the reliability index values (REL_sec) of the prediction (0 = low; 9 = high); H denotes the predicted helical structure. (B) Helical wheel plot of NSP5 residues (178 to 198) predicted to form an α-helical structure. Hydrophobic residues are boxed, and the line divides the helix into a hydrophilic face (face 1) and hydrophobic face (face 2) along the overall hydrophobic moment of the helix (indicated by a solid arrow). Numbers refer to amino acid positions in the RRV NSP5 protein, and the open arrow indicates the residue arrangement along the helix viewed from the top. (C) Predicted structure of the NSP5 C terminus using a fold recognition algorithm. Numbers refer to amino acid positions in the RRV NSP5 protein.

FIG. 3.

Role of C-terminal residues in NSP5 insolubility. (A) Thirteen NSP5 C-terminal mutants were expressed in COS-7 cells, and equivalent amounts of pellet and soluble fractions were analyzed by Western blotting using anti-His antibody as detailed in Materials and Methods. Blots were reprobed for β-actin as an internal control for comparable fractionation and equal loading. For wild-type N-NSP5 (wt) or each of the mutants tested, the S/P ratio was derived (indicated below each protein) as described in Materials and Methods. An asterisk indicates a nonspecific cellular band to demonstrate comparable protein separation for the panels shown. (B) Soluble fraction-pellet fraction distribution of N-NSP5 and proline mutants. The bars represent densitometric values obtained after scanning the blots shown in panel A. NSP5 S/P ratios were normalized to the S/P ratio of actin, internal to each sample and represented as fold increases over the level for wild-type NSP5 protein.

FIG. 4.

Proline substitution of residues in the predicted NSP5 α-helical domain affects the accumulation of hyperphosphorylated isoforms. Proline mutants or wild-type N-NSP5 (N-NSP5 wt) were expressed in COS-7 cells, and cells were exposed to 200 nM of calyculin A (lanes indicated by a plus sign) for 30 min before lysis in buffer containing 2% SDS in order to recover all hyperphosphorylated NSP5 isoforms (37). Basal (b) and hyperphosphorylated (hp) isoforms are indicated by brackets. The asterisk indicates a nonspecific cellular band present in total cell lysates that serves as an internal loading control.

FIG. 5.

Effect of mutations in the α-helix of NSP5 on VLS formation by GFP-C68-NSP5. Cells were transfected with plasmids expressing GFP-NSP5 chimeras, and protein localization was observed 48 h later. (A) Shown are the GFP-NSP5-C68 wild-type protein (left panel), A191M mutant (middle panel), and A191P mutant (right panel). (B) GFP-NSP5-C68 mutants A191P, M192P, and L194P abolish VLS assembly (lower row), whereas A182P, M185P, and Q189P mutant proteins (upper row) do not.

FIG. 6.

VLS formation is regulated by a calcium switch. (A) The GFP-NSP5-C68 protein was expressed in cells, and 24 h later cells were starved of calcium (−Ca²⁺; left panel) as described in Materials and Methods (22, 23). GFP-C68-NSP5 subsequently was monitored 12 min after calcium resupplementation by fluorescence microscopy (+Ca²⁺; right panel). (B) Time-lapse images of rapid VLS formation by GFP-NSP5-C68 following a calcium switch. The left panel shows a field of cells that were starved of calcium as described above. The inset follows GFP-NSP5-C68 fluorescence within a group of cells at 1, 5, and 10 min after exposure to high-calcium medium by time-lapse photography.

FIG. 7.

Effect of helix disruption on the calcium-switch-mediated VLS formation by GFP-NSP5-C68. (A) Wild-type GFP-NSP5-C68 and mutants A182P, M185P, M192P, and L194P were subjected to a calcium switch as described in the legend to Fig. 6. (B) GFP-NSP5-C68 A191 was mutated to either Met or Pro, and the ability of mutants to respond to a calcium switch was visualized by fluorescence microscopy.

FIG. 8.

Identification of functional DxDxD motifs in the NSP5 C terminus. (A) The C terminus of NSP5 contains two tandem DxDxD pseudo-EF-hand calcium motifs (in boldface) upstream of the predicted α-helix. The predicted NSP5 C-terminal α-helix is underlined. Alanine mutations introduced to disrupt DxDxD motifs individually are shown (DXM1 and DXM2), with mutations in boldface. (B) Expression of DXM1 and DXM2 mutant GFP-NSP5-C68 constructs following calcium starvation (upper row) or following exposure to high-calcium medium (lower row).