A single point mutation controls the cholesterol dependence of Semliki Forest virus entry and exit

Abstract

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

Figure 1

Growth of wt and srf mutants in control and sterol-modified C6/36 cells. Wild-type or mutant viruses were prebound on ice for 1 h to control or sterol-modified C6/36 cells at a multiplicity of 1 pfu/cell. Infection was initiated by warming the cells to 28°C for 1.5 h. The cells were then washed to remove input virus, incubation continued at 28°C, and samples were taken at the indicated times for plaque titration on BHK cells. Representative example of two experiments.

Figure 2

Infectivity and fusion of wt and srf-3 in control and cholesterol-depleted C6/ 36 cells. (A) Infection. Control (+) and sterol-depleted (−) cells were infected with serial dilutions of wt and srf-3, and primary infection was quantitated by immunofluorescence. Infection was normalized to 10⁵ infectious centers/ml on control cells. Data shown are the average of 13 determinations. (B) Fusion. Serial dilutions of virus were bound to control (+) or depleted (−) cells in the cold, and the cells treated for 1 min at pH 5.5 to trigger virus fusion with the plasma membrane. Infected cells resulting from virus fusion were quantitated by immunofluorescence, and normalized to 10⁶ infectious centers/ml on control cells. Data shown are the average of six determinations for wt and seven determinations for srf-3.

Figure 3

Cholesterol is required for virus membrane stability. (A) [³⁵S]methionine-labeled wt SFV or srf-3 were prepared by growth in control C6/36 cells. (B) [³⁵S]methionine-labeled srf-3 was prepared by growth in cholesterol-depleted C6/36 cells. Radiolabeled viruses were pelleted, centrifuged on sucrose gradients, the gradients fractionated, and radioactivity determined by scintillation counting. Sedimentation is to the left. Representative example of two experiments.

Figure 4

The growth properties of srf-3 are conferred by the E1 P226S mutation. The growth of wt, srf-3, and viruses derived from the pSP6-SFV-4 infectious clone (wt/ic), pSP6-SFV-4 containing the NdeI fragment of srf-3 (srf-3/ic), and pSP6-SFV-4 containing the P226S mutation was determined on cholesterol-depleted C6/36 cells as in Fig. 1. Representative example of two experiments.

Figure 5

The srf-3 cholesterol-independent infectivity and fusion properties are conferred by the E1 P226S mutation. (A) Infection. Primary infection by viruses derived from the pSP6-SFV-4 infectious clone (wt/ic) and pSP6-SFV-4 containing the P226S mutation was determined on control (+) or cholesterol-depleted (−) C6/36 cells as in Fig. 2, and normalized to 10⁵ infectious centers/ ml on control cells. Average of two experiments. (B) Fusion. Fusion of viruses derived from the pSP6-SFV-4 infectious clone (wt/ic) and pSP6-SFV-4 containing the P226S mutation was determined on control (+) or cholesterol-depleted (−) C6/36 cells as in Fig. 2, and normalized to 10⁷ infectious centers/ ml on control cells. Average of two experiments.

Figure 6

Cholesterol-independent SFV exit is conferred by the E1 P226S mutation. Control or cholesterol- depleted C6/36 cells were infected with the indicated viruses at muliplicities of 50 pfu/cell, except for wt/ic in depleted cells, for which the multiplicity was 1,000 pfu/ cell. 18 h postinfection, the release of progeny virus was detemined by pulse-labeling for 15 min with [³⁵S]methionine and cysteine, chasing for 3 h in the presence of excess cold methionine and cysteine, collecting the chase medium, and lysing the cells. The amount of radiolabeled virus spike proteins in the cells (C) and media (M) was determined by immunoprecipitation and SDS-PAGE. Representative example of two experiments.

Figure 7

Sequence comparison of the P226 E1 region in alphaviruses. Amino acid numbering is given for SFV E1, starting with residue 215, and P226 is shown in bold. Abbreviations and accession numbers are as follows, other sequence references are listed in (Strauss and Strauss, 1994; Kielian, 1995): SFV (Semliki Forest virus); RRV (Ross River virus); ONN (O'Nyong-nyong virus); CHIK (Chikungunya virus) (NCBI 576465); EEE (Eastern equine encephalitis virus); VEE (Venezuelan equine encephalitis virus); AURA (Aura virus); WEE (Western equine encephalitis virus); WHATE (Whataroa virus) (unpublished data from Drs. Y. Shirako and J. Strauss); OCK (Ockelbo virus); SV (Sindbis virus).