The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence - PubMed (original) (raw)

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence

Y E Lu et al. J Virol. 1999 May.

Abstract

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

PubMed Disclaimer

Figures

FIG. 1

Sequence comparison of E1 226 region of SFV, srf-3, SIN, and SIN mutants. The sequence begins at amino acid 215 of the E1 subunit. Dashed lines indicate sequence identity with wt SFV. Mutations from the parental sequence are shown in bold.

FIG. 2

Growth of wt SFV, srf-3, and wt SIN in control and cholesterol-depleted C6/36 cells. Viruses were prebound to either control (A) or cholesterol-depleted (B) C6/36 cells on ice for 1 h at a multiplicity of 1 PFU/cell. Cells were then incubated at 28°C for 2 h to initiate infection, washed to remove input virus, and further incubated at 28°C for the indicated times. Titers of samples of the media were determined by plaque assay on BHK cells. Note that it proved more difficult to remove all input SIN virus inoculum (3-h time point) (A and B) but that no increase in SIN titer was observed in cholesterol-depleted cells (B).

FIG. 3

Infection and fusion of wt SFV, srf-3, and wt SIN in control and cholesterol-depleted cells. (A) Infection. Control (solid bars) and cholesterol-depleted (hatched bars) C6/36 cells grown on coverslips were infected with serial dilutions of SFV, srf-3, and SIN virus stock, and primary infection was quantitated by immunofluorescence. Infection was normalized to 106 infectious centers/ml on control cells. Data are the means of five independent experiments ± standard deviations. (B) Fusion. Serial dilutions of the indicated virus stocks were bound to control (solid bars) and depleted (hatched bars) C6/36 cells on coverslips in the cold for 90 min and then warmed to 28°C in low-pH (either 5.5 or 5.0) medium for 1 min to trigger virus fusion with plasma membrane. The cells infected due to low-pH fusion were quantitated by immunofluorescence, and the titers were normalized to 106 infectious centers/ml on control cells. Data are the means of four independent experiments ± standard deviations.

FIG. 4

Growth of wt SIN,

KN, and

SGM

mutants in control and cholesterol-depleted cells. Control (A) and cholesterol-depleted (B) cells were infected with wt SIN,

KN, and

SGM

viruses at 1 PFU/cell as described in the legend for Fig. 2, and virus titers were measured after growth for the indicated time. Note that the y axes in panels A and B are different.

FIG. 5

Infection and fusion of wt SIN,

KN, and

SGM

mutants in control and cholesterol-depleted cells. (A) Infection. Virus infection of control (solid bars) and depleted (hatched bars) cells was measured as described in the legend for Fig. 3A. Virus titers on control cells were normalized to 107 infectious centers/ml. (B) Fusion. Virus fusion with control (solid bars) and depleted (hatched bars) C6/36 cells was quantitated by immunofluorescence as described in the legend for Fig. 3B. Virus titers on control cells were normalized to 106 infectious centers/ml. Panels A and B show the averages of two independent experiments, and error bars show the ranges.

FIG. 6

Kinetics of exit of wt SIN and

SGM

virus in control and cholesterol-depleted C6/36 cells. (A) Control C6/36 cells were infected with wt SIN or

SGM

at a multiplicity of 10 PFU/cell for 26 h at 28°C. Cells were pulse-labeled for 15 min with [35S]methionine-cysteine at 50 μCi/ml and chased for the indicated times, and aliquots of the media were immunoprecipitated with a monoclonal antibody against E2 in the absence of detergent. The cells were lysed and immunoprecipitated with a polyclonal antibody against the SIN spike protein. The immunoprecipitates were assayed by SDS-PAGE, fluorography, and phosphorimaging, for which 1/20 of the cell lysates and 1/10 of the medium samples were loaded. E1 and E2 comigrate in this gel system. The graph shows the quantitation of virus exit, determined as the percentage of spike proteins in the medium divided by the total amount of spike proteins in the cell lysate at 0 min of chase. Shown is a representative example of two experiments. (B) Cholesterol-depleted C6/36 cells were transfected for 4 h with 2.5 μg of wt SIN RNA or

SGM

RNA plus 10 μg of Cellfectin, incubated in fresh medium at 28°C for 20 h, pulse-labeled for 30 min with [35S]methionine-cysteine at 200 μCi/ml, and chased for the time periods indicated. Analysis was performed as described for panel A, except that for the wt virus, 1/10 of the cell lysates and 1/5 of the medium samples were loaded. Shown is a representative example of three experiments.

FIG. 7

Infection by wt and mutant SIN virus in control and cholesterol-depleted C6/36 cells. Infection of control (solid bars) and cholesterol-depleted (hatched bars) C6/36 cells with wt and mutant SIN virus stocks was quantitated as described in the legend for Fig. 3A. Infection was normalized to 106 infectious centers/ml on control cells. Data are the averages of two independent experiments, and error bars show the ranges.

References

1. Binley J, Moore J P. HIV-cell fusion: the viral mousetrap. Nature. 1997;387:346–348. - PubMed
1. Chatterjee, P., and M. Kielian. 1998. Unpublished results.
1. Chatterjee, P., and M. Kielian. 1998. Unpublished results.
1. Cleverley D Z, Geller H M, Lenard J. Characterization of cholesterol-free insect cells infectible by baculoviruses: effects of cholesterol on VSV fusion and infectivity and on cytotoxicity induced by influenza M2 protein. Exp Cell Res. 1997;233:288–296. - PubMed
1. DeTulleo L, Kirchhausen T. The clathrin endocytic pathway in viral infection. EMBO J. 1998;17:4585–4593. - PMC - PubMed

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence - PubMed (original) (raw)