Structural and functional analysis of the Xestia c-nigrum granulovirus matrix metalloproteinase

Abstract

Sequence analysis of the Xestia c-nigrum granulovirus (XcGV) genome identified an open reading frame encoding a 469-amino-acid (54-kDa) protein with over 30% amino acid sequence identity to a region of about 150 amino acids that includes the catalytic domains of human stromelysin 1 (Str1)/matrix metalloproteinase 3 (MMP-3) (EC 3.4.24.17) and sea urchin hatching enzyme (HE). Stromelysin homologs have not been reported from baculoviruses or other viruses. Unlike human Str1 and sea urchin HE, the putative XcGV-MMP does not have a signal peptide and lacks the peptide motif involved in the cysteine switch that maintains other MMPs in an inactive form. The putative XcGV-MMP, however, possesses a conserved zinc-binding motif in its putative catalytic domain. The XcGV-MMP homolog was cloned, and a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) that expresses XcGV-MMP under the polyhedrin promoter was constructed. A distinct pattern of melanization was observed in B. mori larvae infected with MMP-expressing BmNPV. Fat body extracts from larvae overexpressing the 54-kDa recombinant MMP digested dye-impregnated collagen (Azocoll). The enzymatic activity was inhibited by two metalloproteinase inhibitors, EDTA and 1,10-phenanthroline. These results suggest that the XcGV MMP-3 gene homolog encodes a functional metalloproteinase.

FIG. 1

Amino acid sequence alignment of XcGV-MMP, human Str1, and P. lividus HE using the program Clustal W. The box encloses the consensus zinc-binding motif. The P-R-C-G-(V/N)-P-D sequence, presumably involved in the cysteine switch, is underlined. A hemopexin-like motif and a proline-rich region of Str1 are underlined with double and dotted lines, respectively. White letters within black boxes indicate identical amino acid residues, and shaded boxes denote conserved substitutions. Dashes indicate gaps in the sequence. Amino acid numbers are indicated on the left. The human Str1/MMP-3 sequence and sea urchin HE accession numbers are U78045 and S12805, respectively. The protein motifs were analyzed using the PROSITE motif database.

FIG. 2

B. mori larvae infected with WT or recombinant BmNPVs 24 h after death. The larvae were injected with approximately 2 × 10⁵ PFU of a viral suspension containing 0.6 mg of kanamycin per ml. Mock, mock-infected larvae; WT, BmNPV-infected larvae; 1, BmCysPD-infected larvae; 2, BmMMPCysPD-infected larvae. (A) Dorsal side. (B) Ventral side.

FIG. 3

Coomassie brilliant blue staining (left panel) and Western blot analysis of the XcGV-MMP expressed by BmNPV using antibody against His-tagged rMMP (right panel). Aliquots of FB tissue containing approximately 100 μg of protein were collected from mock- or recombinant BmNPV-infected B. mori larvae and separated by SDS-PAGE. BmMMPCysPD, FB of BmMMPCysPD-infected B. mori larvae 3, 4, 5, and 6 days p.i.; BmCysPD, FB of BmCysPD-infected B. mori larvae collected 6 days p.i.

FIG. 4

Proteinase activity of recombinant XcGV-MMP expressed by BmNPV. (A) Increased A₅₂₀ due to Azocoll digestion with the FB extracts from mock-, BmCysPD-, or BmMMPCysPD-infected B. mori larvae. FB extracts were assayed for proteinase activity in Azocoll solution. (B) MMP activity during infection. FB dissected from mock-infected, BmCysPD-infected (5 days p.i.), or BmMMPCysPD-infected (2, 3, 4, 5, or 6 days p.i.) larvae were assayed for proteinase activity. A 1-mg portion of FB protein was added to the Azocoll solution for the proteinase assay. Error bars indicate the range of triplicate values.

FIG. 5

Ammonium sulfate fractionation of FB extract containing recombinant XcGV-MMP expressed by BmMMPCysPD. (A) Proteinase activity of the FB fractions collected from ammonium sulfate precipitation. 1, FB extract; 2, 0 to 20% saturated ammonium sulfate fraction; 3, 20 to 40% saturated ammonium sulfate fraction; 4, 40 to 60% saturated ammonium sulfate fraction; 5, 60 to 80% saturated ammonium sulfate fraction. (B) Western blot analysis of the ammonium sulfate fractions. The sample numbers in panel A correspond to the sample numbers in panel B. The relative intensities of the bands reacting with MMP antibody were as follows: 1, 185.77; 2, 92.93; 3, 64.97; 4, 47.67; and 5, 19.48 (for the 54-kDa band); and 1, 153.57; 2, 90.11; 3, 13.02; 4, 2.97; and 5, 0 (for the 30-kDa band). FB extract was prepared from FB collected from BmMMPCysPD-infected B. mori larvae 5 days p.i.

FIG. 6

Immunofluorescent staining of BmN cells. The cells were infected with BmMMPCysPD (multiplicity of infection, 10) or mock infected with medium. The cells were immunostained as described in Materials and Methods and examined under a confocal laser-scanning microscope. (A) Transmitted light and immunofluorescence image of mock-infected cells stained with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G polyclonal antiserum (1:200). (B) Image of BmMMPCysPD-infected BmN cells at 36 h p.i., stained as described for panel A. Bar, 10 μm.