Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles

Abstract

Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in the life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.

Fig. 1

Plasma membrane rafts are required for RSV infection. (A) RSV infection of untreated (UT) and methyl-beta-cyclodextrin + lovastatin (MBCD + LOV) treated A549 cells. Viral titer was determined at 24h post-infection by plaque assay. (B) The viral titer values (pfu/ml) shown in Fig. 1A was utilized to calculate percent (%) infection. % infection was calculated based on the ratio of pfu/ml obtained from UT cells vs. MBCD + LOV treated A549 cells. 100% infectivity corresponds to viral titer in UT cells. % infection value represents the mean ± standard deviation for three determinations. (C) VSV infection of UT and MBCD + LOV treated A549 cells. Viral titer was determined at 24h post-infection by plaque assay. (D) RSV infection of UT and MBCD + LOV treated primary normal human bronchial epithelial (NHBE) cells. Viral titer was determined at 48h post-infection by plaque assay. (E) The viral titer values (pfu/ml) shown in Fig. 1D was utilized to calculate percent (%) infection. % infection was calculated based on the ratio of pfu/ml obtained from UT cells vs. MBCD + LOV treated NHBE cells. 100% infectivity corresponds to viral titer in UT cells. % infection value represents the mean ± standard deviation for three determinations. Plaque assay values shown in Figs. 1A, 1C and 1D are expressed as pfu/ml and it represents mean ± standard deviations for three independent determinations. Standard deviations are shown by the error bars.

Fig. 2

Effect of methyl-beta-cyclodextrin (MBCD) on raft structure and cholesterol levels. (A) Fluorescence confocal microscopic analysis of A549 cells labeled with FITC-cholera toxin (CHTX) subunit (green) and DAPI (blue, nucleus) following treatment with MBCD + lovastatin (LOV) (cells incubated with 5 mM MBCD for 1h, followed by addition of lovastatin containing fresh medium for additional 16h). (B) Untreated (UT) and MBCD + LOV (cells incubated with 5 mM MBCD for 1h, followed by addition of lovastatin containing fresh medium for additional 16h) treated A549 cells were used to estimate total cholesterol content by cholesterol quantitation assay kit. Following normalization with protein concentration, the total cholesterol content was expressed as percentage of control. The control cells are UT cells and are represented as 100%. Please see table-1 for the mean ± standard deviation values.

Fig. 3

Rafts are essential for release of infectious RSV particle. (A) Untreated (UT) and methyl-beta-cyclodextrin + lovastatin (MBCD + LOV) treated A549 cells were infected with RSV in the presence of cycloheximide as detailed in the supplemental figure-S2 and in the methods section. Infectious RSV release efficiency was determined by utilizing medium supernatant to assess infectious viral titer by plaque assay. Plaque assay values are expressed as pfu/ml and it represents mean ± standard deviations for three independent determinations. Standard deviations are shown by the error bars. (B) The plaque assay values (pfu/ml) shown in Fig. 3A was utilized to calculate percent (%) infectious RSV release. % infectious RSV release was calculated based on the ratio of pfu/ml obtained from UT cells vs. MBCD + LOV treated cells. 100% infectious RSV release corresponds to viral titer in UT cells. % infectious RSV release value represents the mean ± standard deviation for three determinations.

Fig. 4

Effect of raft disruption on RSV budding and cholesterol content of progeny RSV virion particles. (A) Untreated (UT) and methyl-beta-cyclodextrin + lovastatin (MBCD + LOV) treated A549 cells were infected with RSV as detailed in the figure and in the methods section. After infection, cells were labeled with ³⁵S-methionine to promote budding of radiolabeled RSV particles. Radiolabeled RSV was purified from the medium supernatant of UT and MBCD treated cells. Purified ³⁵S-methionine-RSV was subject to SDS-PAGE and autoradiography to visualize radiolabeled viral proteins. (B) UT and MBCD + LOV treated A549 cells were infected with RSV as detailed in the methods section. RSV was purified from the medium supernatant of UT and MBCD + LOV treated cells. Purified RSV was utilized to estimate cholesterol content by cholesterol quantitation assay kit. Following normalization with protein concentration, the total cholesterol content was expressed as percentage of control virus. The control virus is virion particles obtained from UT cells and is represented as 100%. Please see table-1 for the mean ± standard deviation values.

Fig. 5

RSV infection of cholesterol deficient human fibroblasts - (A) Morphology of mock-infected or RSV-infected (36h post-infection) cholesterol-deficient Niemann-Pick syndrome type C human fibroblasts (NPC cells) and normal human embryonic lung (HEL cells) fibroblasts (wild-type counterpart of NPC cells). (B) RSV infection of NPC and HEL cells. Viral titer was determined at 36h post-infection by plaque assay. (C) The viral titer values (pfu/ml) shown in Fig. 5B was utilized to calculate percent (%) RSV infection. % RSV infection was calculated based on the ratio of pfu/ml obtained from HEL cells vs. NPC cells. 100% infectivity corresponds to viral titer in HEL cells. % infection value represents the mean ± standard deviation for three determinations. (D) VSV infection of NPC and HEL cells. Viral titer was determined at 36h post-infection by plaque assay. Plaque assay values shown in Figs. 5B and 5D are expressed as pfu/ml and it represents mean ± standard deviations for three independent determinations. Standard deviations are shown by the error bars.

Fig. 6

Release of infectious RSV particle is disrupted in cholesterol deficient human fibroblasts. (A) HEL and NPC cells were infected with RSV in the presence of cycloheximide as detailed in the supplemental figure-S4B and in the methods section. Infectious RSV release efficiency was determined by utilizing medium supernatant to assess infectious viral titer by plaque assay. Plaque assay values are expressed as pfu/ml and it represents mean ± standard deviations for three independent determinations. Standard deviations are shown by the error bars. (B) The plaque assay values (pfu/ml) shown in Fig. 6A was utilized to calculate percent (%) infectious RSV release from NPC and HEL cells. % infectious RSV release was calculated based on the ratio of pfu/ml obtained from HEL cells vs. NPC cells. 100% infectious RSV release corresponds to viral titer in HEL cells. % infectious RSV release value represents the mean ± standard deviation for three determinations.