Novel form of fibronectin from zebrafish mediates infectious hematopoietic necrosis virus infection

Abstract

The presence of a novel form of zebrafish fibronectin (FN2) on the cell surface increased the cell's susceptibility to infection by infectious hematopoietic necrosis virus (IHNV). Unlike other fibronectins, FN2 possesses a truncated structure and accumulates on the cell surface instead of in the extracellular matrix. Fish embryo cells expressing recombinant FN2 were more susceptible to IHNV infection, with a greater percentage of cells exhibiting cytopathic effect (CPE) compared to nontransfected control cells. Incubation of nontransfected cells with soluble recombinant FN2 increased IHNV infection, as measured by plaque assay. The number of plaques increased in correlation with the amount of protein added and the length of time that cells were incubated with the protein. Incubation of IHNV with soluble FN2 before addition to cells also increased infection. FN2 immobilized on the culture surface inhibited IHNV infection. The results indicate that FN2 present on the cell surface is able to mediate IHNV attachment and cell entry.

FIG. 1.

(A) Schematic diagram showing the organization of zebrafish FN1 and FN2 (45). The type I (○), II (•), and III (□) repeats along with EIIIA, EIIIB, and the variable region (V) are shown. The amino acid sequence of the FN2 C-terminal tail is indicated. (B) Cellular distribution of recombinant FN1 and FN2, each synthesized as a fusion protein with GFP in CHSE-214 cells. Photomicrographs of the same field examined by light (left) and UV (right) phase-contrast microscopy showing the distribution of the GFP-labeled proteins. Magnification, ×200.

FIG. 2.

Expression of recombinant FN2 by CHSE-214 cells. FN2 was detected by Western blot analysis of proteins present in medium taken from a 7-day-old culture of CHSE-214 cells expressing the pBK-RSV-FN2 plasmid (lane 3). Although recombinant FN2 initially localizes on the cell surface, it accumulates in the medium after several days of constitutive expression. Nontransfected CHSE did not produce detectable amounts of FN2 (lane 2). Molecular size markers are shown in lane 1.

FIG. 3.

Phase-contrast photomicrograph showing nontransfected (A) and FN2-transfected (B) CHSE-214 cells 1, 2, and 3 days following IHNV infection (10⁻²). Individual cells exhibiting CPE initially appear round and translucent. By day 3, 100% of the FN2-transfected cells exhibit CPE, indicated by aggregates of rounded cells detaching from the culture surface, while a small percentage of cells in the control culture are just beginning to exhibit CPE. Magnification, ×144.

FIG. 4.

Electron photomicrograph showing the presence of IHNV particles (arrow) in a CHSE-FN2 culture 1 day following exposure to the virus. Virus particles were not detected in a nontransfected CHSE-214 culture (left) exposed to IHNV on the same day. Magnification, ×7,800.

FIG. 5.

IHNV titer in medium obtained from FN2-transfected (solid bars) and nontransfected (open bars) CHSE cells. Medium was collected from each culture 1, 2, or 3 days following IHNV infection, and virus titer was measured by plaque assay as described in Materials and Methods. The mean titer of triplicate samples is shown with the standard error.

FIG. 6.

Effect of exogenously added recombinant FN2 on IHNV infection. (A) CHSE-214 cells were incubated (30 min, room temperature) with soluble recombinant FN2 at the indicated concentration before being exposed to IHNV (10⁻⁵). The number of plaques produced in the culture was determined as described in Materials and Methods. (B) CHSE-214 cells were incubated with soluble recombinant FN2 (10 μg/ml) for the length of time indicated before being exposed to IHNV (10⁻⁵), and the number of plaques produced was determined. (C) IHNV (10⁻⁵) was incubated with the indicated concentration of soluble recombinant FN2 before being added to CHSE-214 cells, and the number of plaques produced in the culture was determined. For each experiment, the mean number of plaques produced in two cultures along with the standard error is shown. Plaque numbers that are significantly different from the control, determined by the Duncan’s multiple-range test (α = 0.05), are indicated (*). (D) Photograph showing plaque assays that were performed on CHSE-214 cultures grown on plates containing immobilized FN2 at the indicated concentration.