Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo

Abstract

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.

FIG. 1

Ribbon diagram showing a side view (A) and a top view (B) of the ectodomain portion of the TBE virus protein E homodimer (55). The positions of mutations generated by passaging the virus in BHK-21 cells are shown by white spheres, with the amino acid numbers indicated. Domain I of each subunit is colored red, domain II is yellow, and domain III is blue. The fusion peptide (2) at the tip of domain II is colored green, and the disulfide bridges are orange. The first N-acetyl-glucosamine residue of the carbohydrate attached to Asn 154 is shown in gray, and the position where the peptide chain continues into the stem-anchor region at the carboxy terminus is indicated by a “C.”

FIG. 2

Surface models of wild-type and mutant E proteins colored by electrostatic potential. The viewing angle is the same as in Fig. 1B. Positively charged surfaces are shown in blue, and negatively charged surfaces are red. The yellow ovals indicate areas of increased positive charge relative to wild-type protein E.

FIG. 3

Infectivity titers in BHK-21 (open bars) and CE (solid bars) cells. Titers were determined by endpoint dilution experiments (see Material and Methods), starting with 10⁴ PFU of each virus. Values shown are the means of four independent experiments (with error bars representing standard deviations).

FIG. 4

Growth curves obtained by infecting BHK-21 cells grown under normal conditions (●) or under conditions that prevent sulfation of proteoglycans (○). Cells were infected with 10 infectious units of wild-type or recombinant mutant virus, and release of virus into the supernatants was monitored by a protein E ELISA (for details, see Materials and Methods). A representative example of several experiments is shown. hpi, hours postinfection; OD, optical density.

FIG. 5

Inhibition of virus growth by soluble heparin. Heparin concentrations added to the virus and growth medium are as follows: ●, no heparin; ○, 50 μg/ml; □, 150 μg/ml; ∗, 500 μg/ml. Cells were infected with 10 infectious units of wild-type or recombinant mutant virus, and release of virus into the supernatants was monitored by a protein E ELISA (for details, see Materials and Methods). A representative example of several experiments is shown. hpi, hours postinfection; OD, optical density.

FIG. 6

Binding of wild-type and mutant viruses to BHK-21 cells. Equal amounts (500 ng) of purified virus were added to undigested BHK-21 cells (−) or cells that were predigested (+) with heparinase III (top) or chondroitinase ABC (bottom). The amount of cell-bound virus was quantified by fluorescence-activated cell sorter analysis (see Materials and Methods). Median fluorescence intensities are plotted as arbitrary units (a.u.). A representative example of several experiments is shown.

FIG. 7

Virulence (neuroinvasiveness) and peripheral infectivity of wild-type and recombinant mutant viruses determined by subcutaneous inoculation of 5-week-old mice. LD₅₀s (top) and ID₅₀s (middle) were determined as described in Materials and Methods. The attenuation index (bottom) was calculated as the LD₅₀/ID₅₀ ratio.