Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor - PubMed (original) (raw)

Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor

W B Klimstra et al. J Virol. 1998 Sep.

Abstract

Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981-1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.

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Figures

FIG. 1

FIG. 1

Survival of neonatal CD-1 mice infected with TR339 (solid line; n, 30; AST, 1.8 ± 0.34 days), TRSB (evenly dashed line; n, 30; AST, 3.2 ± 0.46 days), or TRSB-R114 (unevenly dashed line; n, 25; AST, 7.9 ± 2.5 days). Mice were inoculated subcutaneously with 1,000 BHK PFU of virus in 50 μl of diluent.

FIG. 2

FIG. 2

Relative binding and specific infectivity of TR339, TRSB, and TRSB-R114 for BHK cells. For binding assay (solid bars), 105 cpm of each radiolabeled virus was added to 106 BHK cells in suspension and incubated at 4°C for 60 min. After three washes with VB, radioactivity associated with cells was quantitated. For specific infectivity (hatched bars), radiolabeled virus was diluted and assayed for plaque formation on BHK cell monolayers. Specific infectivity (PFU/cpm) was then calculated for each virus. Values are presented as percentages of values for TRSB (100%). ∗, binding of TR339 was <1% of that of TRSB in this assay.

FIG. 3

FIG. 3

Soluble heparin competition of TR339, TRSB, TRSB-R114, and Toto 50. (A) Reduction in plaque formation of TR339 (squares), TRSB (circles), TRSB-R114 (triangles), and Toto 50 (diamonds) on BHK cell monolayers with increasing concentrations of heparin. Viruses were diluted to 100 to 200 PFU/200 μl and reacted with heparin for 30 min at 4°C, followed by infection of BHK cell monolayers in the presence of heparin. (B) Reduction in binding of TR339 (squares), TRSB (circles), and TRSB-R114 (triangles) to BHK cells with increasing concentrations of heparin. Viruses (105 cpm/reaction) were reacted with heparin as described above, followed by binding assay in the presence of heparin. Results are averages of two or three reactions at each concentration.

FIG. 4

FIG. 4

Effect of heparinase digestion on plaque-forming efficiency of TR339, TRSB, and TRSB-R114 on BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with virus buffer and infection with 100 to 200 PFU of TR339 (squares), TRSB (circles), or TRSB-R114 (triangles). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III, washed, and infected as above. Data are averages of two or three assays at each concentration.

FIG. 5

FIG. 5

Effect of heparinase digestion on binding of TRSB (circles) and TRSB-R114 (triangles) to BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with VB, suspension, and use in binding assays (5 × 104 cpm of radiolabeled virus per reaction). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III and processed as above. Data are averages of two or three binding reactions at each concentration. In these assays, binding of TR339 to digested or undigested cells was not above background cpm measured in cell-free control reactions.

FIG. 6

FIG. 6

Infectivity of TR339, TRSB, and TRSB-R114 GFP-expressing RP and binding of analogous viruses to wild-type CHO-K1 cells and CHO cell mutants deficient in all GAGs (pgsD-745) or HS (pgsD-677). (A) Monolayers of each cell type were infected (60 min) with 100 to 200 BHK GFP-infectious units followed by three washes with VB, medium replacement, and incubation for 8 to 12 h. Cells expressing GFP were enumerated by fluorescence microscopy. Data are averages of four infections of TR339 (hatched bars), TRSB (solid bars), and TRSB-R114 (cross-hatched bars). (B) Suspension binding assays (105 cpm added per reaction) were completed as described for BHK cells. Results for TR339 (hatched bars), TRSB (solid bars), and TRSB-R114 (cross-hatched bars) are presented as percent cpm bound and represent averages of two or three reactions.

FIG. 7

FIG. 7

Changes in titer, heparin competition, BHK cell binding, and heparin-agarose bead binding phenotypes of TR339 after passage in BHK cells. (A) BHK cell monolayers were infected at a multiplicity of infection of 1 followed by incubation for 20 h, harvesting of supernatants, and infection of new monolayers. This cycle was repeated for a total of five passages. Hatched bars (left y axis) indicate BHK cell titers for each of the five passages. The solid line (right y axis) indicates change in inhibition of BHK cell plaque-forming efficiency due to competition with soluble heparin (200 μg/ml). (B) Increases in BHK cell binding (solid bars) and specific infectivity (hatched bars) of virus derived from the third passage, compared to TR339 stock virus. (C) Increase in heparin-agarose bead binding (solid bars) but not BSA-agarose bead binding (hatched bars) of third-passage virus, compared to TR339 stock virus. ∗, binding of either virus to BSA-agarose beads was <1% of added cpm.

FIG. 8

FIG. 8

Identification of heparin interaction consensus sequences (from reference 7) in the PE2 glycoprotein. Matches (XBBXBX or XBXBBX) are indicated in boldface type within the glycoprotein sequence (residue numbers of the amino-terminal consensus match residues are in boldface type below the sequence). E2 1, E2 70, and E2 114 mutations are indicated in boldface type above the glycoprotein sequence. The site of furin protease cleavage of PE2 is indicated by the arrow. Sequence matches adjacent to the transmembrane domain have not been included.

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