Heparan sulfate facilitates Rift Valley fever virus entry into the cell - PubMed (original) (raw)
Heparan sulfate facilitates Rift Valley fever virus entry into the cell
S M de Boer et al. J Virol. 2012 Dec.
Abstract
Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.
Figures
Fig 1
RVFVns infection is drastically reduced in the absence of GAGs. The CHO 15B and CHO 745 mutant cells derived from the CHO K1 cell line and the CHO lec1 and CHO lec2 mutant cells derived from the CHO Pro5 cell line were cultured in Ham's F-12K medium (Invitrogen) supplemented with 10% fetal calf serum (FCS). Subconfluent monolayers were infected with RVFVns at different multiplicities of infection (MOIs) (0.12 and 0.6). At 20 h postinfection (p.i.), the cells were washed once with phosphate-buffered saline (PBS) and prepared for fluorescence microscopy (A) or fluorescence-activated cell sorter (FACS) analysis (B). (A) Cells were fixed with 3.7% formaldehyde–PBS for 20 min at room temperature, and representative pictures were taken using an Evos fl microscope (AMG) (magnification, ×4; data shown refer to infections at an MOI of 0.6). Nuclei were counterstained with 4′,6′diamidino-2-phenylindole (DAPI). MOCK, mock-infected cells. (B) Cells were trypsinized and fixed with 3.7% formaldehyde–PBS for 20 min at room temperature, and RVFVns-infected (GFP-positive) cells were quantified by FACS (FACSCalibur). Graphical data shown are normalized to the infectivity of CHO K1 or CHO Pro5 cells and are representative of the results of two independent experiments performed in triplicate. Significant differences between conditions are indicated (analysis of variance [ANOVA]-Bonferroni); ***, P < 0.001. Error bars represent standard deviations (SD).
Fig 2
RVFVns infection is decreased in the presence of heparin and after enzymatic removal of heparan sulfate from the cell surface. (A) RVFVns and VSVns were incubated with different concentrations of soluble heparin (MPBio) for 10 min at room temperature in culture medium, prior to infection of CHO K1 cells. At 8 (VSVns) or 20 (RVFVns) h p.i., infection was quantified by FACS analysis as described for Fig. 1. The data shown correspond to the results of a representative set of two independent experiments performed in triplicate. (B) GAGs were enzymatically removed from the cell surface of CHO K1 cells. Chondroitinase ABC (specific for chondroitin and dermatan sulfate), heparinase I (specific for heparin and highly sulfated domains), heparinase II (specific for heparin and heparan sulfate), and heparinase III (specific for heparan sulfate), all purchased at Sigma, were dissolved in resuspension buffer (20 mM HEPES [pH 7.5], 50 mM NaCl, 4 mM CaCl2, 0.01% bovine serum albumin [BSA]). Dilutions were prepared in digestion buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 4 mM CaCl2, 0.1% BSA). CHO K1 cells were treated for 1 h at 37°C with heparinase I, II, or III, with a combination of them, or with chondroitinase at the indicated concentrations. The cells were washed twice with culture medium and then incubated with RVFVns or VSVns for 30 min at 37°C. The cells were washed twice with culture medium and further incubated in culture medium at 37°C for 8 (VSVns) or 20 (RVFVns) h, after which infection was quantified by FACS analysis as described for Fig. 1. Data were obtained from two independent experiments performed in duplicate. Significant differences between conditions are indicated (ANOVA-Bonferroni); ***, P < 0.001. Error bars represent SD.
Fig 3
RVFVns infection strongly depends on sulfation of heparan sulfate. (A) CHO K1 cells were subjected to two passages in culture medium containing 50 mM NaClO3 (Sigma) and subsequently cultured in the presence of 50 mM sodium chlorate, or in chlorate-free culture medium for 30 or 8 h prior to infection, to reverse the chlorate effect. Twenty h postinfection, cells were analyzed by fluorescence microscopy or FACS analysis as described for Fig. 1. Graphical data shown are normalized and are representative of the results of two individual experiments performed in triplicate. (B) A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (FCS). CHO K1 or A549 cells were subjected to two passages in culture medium containing 50 mM NaClO3 (Sigma) and subsequently cultured in the presence of 50 mM sodium chlorate [NaClO3 (+)] or in chlorate-free culture medium [NaClO3 (−)] for 8 h prior to inoculation with RVFVns or VSVns at the indicated MOI. At 8 (VSVns) or 20 (RVFVns) h p.i., cells were analyzed by fluorescence microscopy or FACS analysis as described for Fig. 1. Significant differences between conditions are indicated (ANOVA-Bonferroni; *** = P < 0.001). Error bars represent SD.
Fig 4
Entry of RVFVGFP into GAG-deficient CHO cells is inefficient due to the lack of heparan sulfate. Mutant CHO pgsD-677 cells (HS[−], able to express all GAGs except for heparan sulfate) and pgsA-745 cells (CHO GAG[−], deficient in expression of all GAGs) and the parental CHO K1 cells were inoculated with RVFVns, VSVns, or RVFVGFP. At 8 (VSVns), 10 (RVFVGFP), or 20 (RVFVns) h p.i., cells were analyzed by fluorescence microscopy and GFP-expressing RVFV-infected cells were quantified. Graphical data shown are normalized to the infectivity of CHO K1. Significant differences between conditions are indicated (ANOVA-Bonferroni); ***, P < 0.001. Error bars represent SD.
References
- Baeuerle PA, Huttner WB. 1986. Chlorate—a potent inhibitor of protein sulfation in intact cells. Biochem. Biophys. Res. Commun. 141:870–877 - PubMed
- Cardin AD, Weintraub HJ. 1989. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis 9:21–32 - PubMed
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