PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Virology. Author manuscript; available in PMC Jan 20, 2013.
Published in final edited form as:
PMCID: PMC3249476
NIHMSID: NIHMS336291
Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles
Te-Hung Chang, Jesus Segovia, Ahmed Sabbah, Victoria Mgbemena, and Santanu Bose*
Department of Microbiology Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, U.S.A
* Corresponding author: Santanu Bose, Ph.D., Department of Microbiology & Immunology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC-7758, San Antonio, TX 78229, Tel: (210) 567 1019, FAX: (210) 567 6612, bose/at/uthscsa.edu
Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in 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.
Keywords: Respiratory syncytial virus, cholesterol, lipid rafts, virus release
Paramyxoviruses are enveloped viruses containing a non-segmented negative strand single-stranded RNA genome. Paramyxoviruses are important human pathogens known to cause many diseases. All paramyxoviruses enter cells via direct fusion of the viral lipid-envelope with the plasma membrane (non-endocytic pathway) and viral assembly/budding occurs in the host plasma membrane. Human respiratory syncytial virus (RSV) is a lung-tropic paramyxovirus that causes high morbidity and mortality among infants, children, and the elderly (Collins et al., 2007; Hall, 2001; Hippenstiel et al., 2006). RSV infects the airway to cause respiratory diseases like pneumonia and bronchiolitis.
Paramyxoviruses like RSV utilize a wide spectrum of cellular proteins during its life cycle. Plasma membrane assembly and release (budding) of paramyxoviruses is an essential component of viral life cycle. Several cellular proteins were shown to assist this stage of viral replication (Gower et al., 2005; Ravid et al., 2010; Utley et al., 2008). Lipid raft micro-domains in the plasma membrane are enriched with cholesterol and sphingolipids (Pike, 2003, Silvius, 2003). Due to the presence of cholesterol, the raft domain membrane possesses an “ordered” rigid structure with limited “fluidity” compared to the surrounding plasma membrane. Lipid rafts serve as platforms for plasma membrane assembly and budding of enveloped viruses like influenza A virus (Leser and Lamb, 2005; Scheiffele et al., 1999; Takeda et al., 2003; Xiangjie and Whittaker, 2003), Sendai virus (Ali and Nayak, 2000), measles virus (Ayota et al., 2004; Manié et al., 2000; Robinzon et al., 2009; Vincent et al., 2000), Newcastle disease virus (NDV) (Dolganiuc et al., 2003; Laliberte et al., 2006; Laliberte et al., 2007). In addition, several enveloped viruses (e.g. Borna disease virus, Ebola virus, Marburg virus) require intact cell-surface lipid rafts for efficient cellular entry (Bavari et al., 2002; Clemente et al., 2009). Although several studies have reported targeting of paramyxovirus proteins to rafts (Ali and Nayak, 2000; Ayota et al., 2004; Dolganiuc et al., 2003; Laliberte et al., 2006; Laliberte et al., 2007; Manié et al., 2000; Robinzon et al., 2009; Vincent et al., 2000), only a few have demonstrated the functional requirement of cell surface rafts for paramyxovirus infection. Assembly of NDV (an avian paramyxovirus) proteins occurs in raft domains and raft is required for release of infectious NDV particles (Dolganiuc et al., 2003; Laliberte et al., 2006; Laliberte et al., 2007). Parainfluenza virus 5 (a paramyxovirus that was formerly known as simian virus 5 or SV5) also requires caveolin (a protein component of caveolae which similarly to lipid rafts is enriched with cholesterol and sphingolipids) for assembly/budding (Ravid, et al., 2010). Several studies reported that – a) RSV proteins (envelope proteins, matrix protein, polymerase proteins) are localized in raft domains (Brown et al., 2002; Brown et al., 2004; Fleming et al., 2006; Marty et al., 2004; McDonald et al., 2004; Oomens et al., 2006), and b) purified RSV virion particles contain raft associated cellular proteins due to incorporation of rafts in the virion envelope during budding process (Brown et al., 2004; Marty et al., 2004; Yeo et al., 2009). Although these studies have suggested raft’s involvement during RSV infection, the exact role of rafts during infection is not known. Moreover, the role of raft during RSV infection of human lung epithelial cells (the cells productively infected by RSV during infection of the respiratory tract) has not been investigated since previous studies were primarily conducted in HEp-2 (a human laryngeal cell-line contaminated with Hela, a human cervical cell-line) and Vero (African Green monkey kidney cells) cells (Brown et al., 2002; Brown et al., 2004; Fleming et al., 2006; Marty et al., 2004; McDonald et al., 2004; Oomens et al., 2006; Yeo et al., 2009).
In the current study we have examined the role of rafts during RSV infection of human lung epithelial cells and human fibroblasts lacking cell-surface raft domains. Our studies revealed that rafts play a role during RSV life-cycle, since they are critical for release of infectious progeny virion particles from infected cells. Disruption of rafts (by the raft disrupting agent methyl-beta-cyclodextrin or MBCD) in human lung epithelial cells resulted in drastic reduction in RSV infection. Raft disruption during infection culminated in release of progeny virion particles with significantly reduced infectivity. Our studies with lung epithelial cells were further validated by utilizing Niemann-Pick syndrome type C human fibroblasts (NPC) [these cells lack normal rafts as a result of defective cholesterol trafficking to the plasma membrane (Ikonen and Holtta-Vuori, 2004; Koike et al., 1998; Laliberte et al., 2007)] and normal human embryonic lung (HEL) fibroblasts (wild-type counterpart of NPC cells) (Laliberte et al., 2007). Our studies with these cells revealed significant reduction in RSV infectivity in NPC cells compared to HEL cells. Further characterization demonstrated that reduced infectivity is due to failure of NPC cells to efficiently release infectious RSV virion particles. Thus, our studies have demonstrated that – a) intact plasma membrane rafts are required for RSV infection, and b) cell surface rafts are critical for release of infectious progeny RSV particles.
Effect of raft disruption on RSV infectivity
Methyl-beta-cyclodextrin (MBCD), a cholesterol-extracting agent has been extensively utilized to disrupt lipid rafts in the plasma membrane (Allsopp et al., 2010; Laliberte et al., 2006; Laliberte et al., 2007; Medigeshi et al., 2008; Xiangjie and Whittaker, 2003; Xu et al., 2009). In order to study the role of rafts during RSV infection, we treated human lung epithelial A549 cells with MBCD. A549 cells are airway cells that are routinely used as a model of type II alveolar epithelial cells. After 1.5h adsorption (at 37°C) of A549 cells with RSV (0.2 MOI), cells were washed and fresh medium was added in the presence of 5mM of MBCD. After 1h incubation with MBCD after virus adsorption, fresh medium containing lovastatin (4 µg/ml) was added. The rationale for adding lovastatin is to inhibit cholesterol bio-synthesis so that cell surface devoid of cholesterol (i.e. loss of cholesterol-rich lipid rafts) is maintained during the course of infection. After 24h post-infection, medium supernatant was collected to determine viral titer by plaque assay analysis. Treatment of A549 cells with MBCD resulted in significant decrease in RSV infectivity (inhibited by 85%–90%) compared to untreated cells (Fig. 1A and 1B). The effect of MBCD is specific for RSV, since similar MBCD treatment of A549 cells did not alter Vesicular Stomatitis Virus (VSV) infection (Fig. 1C).
Fig. 1
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 (more ...)
We next evaluated the requirement of raft during RSV infection of primary normal human bronchial epithelial (NHBE) cells. Similar to A549 cells, NHBE cells were incubated with RSV (0.2 MOI) for 1.5h. After adsorption, fresh medium containing 5 mM MBCD was added to washed cells. Following 1h MBCD treatment, cells were washed and incubated with lovastatin. At 48h post-infection, medium supernatant was collected to determine RSV titer by plaque assay analysis. Cell surface rafts are also required for RSV infection of NHBE cells, since drastic reduction (by 95%) in viral infectivity was observed in MBCD treated cells, compared to control (MBCD untreated) cells (Fig. 1D and 1E). The experimental procedure for the above mentioned studies are presented schematically in supplemental Fig. S1A and S1B.
Previously 5mM-10mM MBCD was used to disrupt cell surface or virus associated raft domains(Allsopp et al., 2010; Laliberte et al., 2006; Laliberte et al., 2007; Medigeshi et al., 2008; Xiangjie and Whittaker, 2003; Xu et al., 2009). In order to examine raft status in A549 cells, cells (untreated and MBCD treated) were incubated with FITC conjugated cholera-toxin subunit-B (CHTX), which specifically binds to the cell surface raft resident ganglioside GM1 (Harder et al., 1998). Based on the GM1 binding property of CHTX, it has been widely used to visualize lipid-rafts on the cell surface (Harder et al., 1998). A549 cells were incubated with 5 mM MBCD for 1h. The cells were then washed and fresh medium containing lovastatin (4 µg/ml) was added. At 16h post-treatment with lovastatin, cells were incubated with FITC-CHTX. FITC-CHTX incubated cells were fixed and visualized by confocal microscopy. As shown in Fig. 2A, while a cell surface ring representing the plasma membrane rafts were prominent in untreated cells, MBCD treatment led to disappearance of the raft structures. Interestingly, MBCD treatment led to intracellular clustering of GM1. The ability of MBCD to reduce cholesterol levels in A549 cells was also investigated. A549 cells were treated with MBCD and lovastatin as described above for FITC-CHTX labeling studies. The loss of cellular cholesterol levels following MBCD treatment was also evident from presence of significantly less cholesterol in MBCD treated A549 cells compared to untreated cells (Fig. 2B) (Table-1). Similar to A59 cells, we also observed loss of cell surface rafts and significant reduction in cellular cholesterol levels in NHBE cells treated with 5 mM MBCD for 1h (data not shown). Treatment of A549 and NHBE cells with 5 mM MBCD (for 1h) and lovastatin (for 24h-48h) was not toxic during the time-frame of our experiment as deduced by trypan blue exclusion viability assay (data not shown). Please note that similar toxicity assays were performed for all the experiments described in Fig. 1. Our studies have demonstrated that rafts are critical for RSV infection of both human alveolar epithelial (A549 cells) cells and primary human bronchial epithelial (NHBE cells) cells.
Fig. 2
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 (more ...)
Table 1
Table 1
Cholesterol estimation of A549 cells and purified RSV virion particles.
Intact rafts are critical for release of infectious RSV particles
Rafts may play an important role during post-entry stages of RSV life-cycle, since addition of MBCD following adsorption inhibited virus infection (Fig. 1). We speculated that rafts may be required for RSV release/budding because assembly/budding of numerous enveloped viruses occurs in the raft domains of the plasma membrane. Moreover, RSV proteins accumulate in the raft domain of infected cells. In order to examine this possibility, we performed an infectious virus release/budding assay as described previously (Bose et al., 2001). A549 cells were infected with RSV (1 MOI) for 8h and then treated with MBCD (5 mM for 1h). After washing, fresh medium was added in the presence of lovastatin (4 µg/ml) and cycloheximide (30 µg/ml cycloheximide was added to accumulate an intracellular viral protein pool in the absence of de novo protein synthesis.). Following 3h incubation, cells were washed 3X and once again fresh medium (+/− lovastatin, but with no cycloheximide) was added to the cells to examine the release/budding of the accumulated viral proteins as virion particle in the presence of intact (cells not incubated with MBCD and lovastatin) and disrupted (MBCD and lovastatin treated cells) rafts. After 12 h the medium supernatants were assessed for viral titer by plaque assay. The budding assay revealed that loss of rafts resulted in diminished release of infectious RSV by 80%–85% (Fig. 3A and 3B). The experimental procedure for the studies described in Figure-3 is presented schematically in supplemental Fig. S2
Fig. 3
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 (more ...)
Inhibition of RSV release from raft disrupted cells could be due to several reasons – a) abnormal assembly of viral proteins occurs in raft disrupted cells, resulting in budding blockade; in this case progeny virion particles will not be present in the medium supernatant, b) normal assembly/budding occurs from raft disintegrated cells, but the progeny virion particles are defective and non-infectious probably due to lack of viral proteins (especially envelope proteins that are required for cellular entry), c) progeny virion particles possess viral proteins, but the particle is non-infectious since it is devoid of virion envelope-associated rafts, which is required for initiating subsequent infection.
In order to examine these possibilities, we studied viral protein composition of purified virions obtained from the medium supernatants of infected cells. These studies were performed essentially similar to the budding assay (+/− MBCD as described above for Fig. 3), but the cells were labeled with 35S-methionine and the experiment was performed in a larger scale (to recover larger amount of purified virus for analysis). The radioactive medium supernatant obtained from untreated and MBCD treated cells was utilized to purify 35S-methionine labeled RSV (35S-RSV) particles. Purified 35S-RSV was then subjected to SDS-PAGE and autoradiography. As shown in Fig. 4A, disruption of rafts did not result in inhibition of assembly/budding of virion particles, since virions derived from both untreated and MBCD treated cells comprised similar levels of viral proteins. Protein assay analysis also did not demonstrate any significant difference in total protein content of purified virions obtained from untreated cells vs. MBCD treated cells (data not shown). The experimental procedure for the studies described in Figure-4A is presented schematically in supplemental Fig. S3.
Fig. 4
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 (more ...)
Although the progeny virions recovered from the medium of untreated and MBCD treated cells had similar protein composition, we examined the cholesterol content of the purified virion particles. As described above, medium supernatant from untreated and MBCD treated ells were utilized to purify RSV particles. The total cholesterol content of these purified virion particles were assayed. The cholesterol content of purified RSV particles obtained from MBCD treated cells were significantly less compared to particles obtained from untreated cells (Fig. 4B) (Table-1). These results suggested that cell surface raft serve as a platform for release of infectious RSV virion particles from infected cells. Moreover, raft is required for enrichment of cholesterol in the RSV virion particles.
RSV infection of cells deficient in plasma membrane cholesterol
In the above studies we have utilized MBCD to disrupt raft micro-domains. In order to provide further evidence for the role of rafts during RSV infection, we utilized Niemann-Pick syndrome type C human fibroblasts (NPC cells). NPC cells are human fibroblasts that harbor mutations in either NPC1 and/or NPC2 genes (Ikonen and Holtta-Vuori, 2004; Koike et al., 1998; Laliberte et al., 2007). Due to these mutations, cholesterol trafficking to the plasma membrane in these cells is defective and as a consequence these cells do not possess functional plasma membrane lipid rafts. These cells have been widely used to study the role of rafts in various cellular processes (Henderson et al., 2000), including its role during virus (e.g. NDV, HIV-1) infection (Laliberte et al., 2007; Tang et al., 2009).
RSV infection of NPC and HEL [normal human embryonic lung (HEL) fibroblasts were used as the wild-type counterpart to compare with mutant NPC cells (Laliberte et al., 2007; Tang et al., 2009)] cells revealed significantly diminished cytopathic effect of RSV upon infection of NPC cells compared to HEL cells (Fig. 5A). The observed cytopathic effect in HEL could be due to enhanced syncytia in HEL cells compared to NPC cells. Since syncytia occur due to cell-cell fusion mediated by RSV fusion protein, it reflects the infection efficiency. Thus, enhanced cytopathic effect (due to syncytia) observed in HEL cells suggested enhanced infectivity of these cells compared to NPC cells. In order to determine whether reduced cytopathic effect in cholesterol deficient NPC cells was due to reduced RSV infectivity, we infected HEL and NPC cells with RSV (0.5 MOI). At 36h post-infection, medium supernatant was collected to determine RSV titer by plaque assay analysis. Indeed, RSV infection was drastically reduced (by 90%) in NPC cells compared to HEL cells (Fig. 5B and 5C). In contrast, no significant difference in infectivity was observed following infection of HEL and NPC cells with VSV (Fig. 5D). The experimental procedure for the studies described in Figure-5B is presented schematically in supplemental Fig. S4A.
Fig. 5
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) (more ...)
We speculated that reduced RSV infectivity in NPC cells could be as a consequence of defective release of infectious RSV particles, since rafts play an essential role during this process (Fig. 3). Therefore, we performed virus release/budding assay as described above for A549 cells. HEL and NPL cells were infected with RSV (1 MOI). At 10h post-infection, cells were washed and the medium was replaced with fresh medium containing cycloheximide (30 µg/ml) to accumulate an intracellular viral protein pool in the absence of de novo protein synthesis. Following 3h incubation with cycloheximide, cells were washed 3X and once again fresh medium (in the absence of cycloheximide) was added to the cells to promote release/budding of the accumulated viral proteins as virion particles. After 12h, the medium supernatants were assessed for viral titer by plaque assay. Similar to lung epithelial cells, we also observed defective release of infectious RSV particle from cholesterol lacking NPC cells compared to normal HEL cells (Fig. 6A and 6B), although viral protein levels in the purified virions derived from NPC and HEL cells were similar (data not shown). Moreover, cholesterol content of NPC cell derived virion particles was significantly less compared to purified virus obtained from HEL cells (data not shown). The experimental procedure for the studies described in Figure-6 is presented schematically in supplemental Fig. S4B. Thus, our studies have demonstrated that plasma membrane cholesterol rich raft domain is required for RSV infection, by virtue of its role in release of infectious progeny RSV particles from infected cells.
Fig. 6
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 (more ...)
Plasma membrane resident cholesterol and sphingolipid enriched lipid rafts play an important role in cellular entry and assembly/morphogenesis of both segmented and non-segmented negative-sense ssRNA viruses (Ali and Nayak, 2000; Ayota et al., 2004; Bavari et al., 2002; Clemente et al., 2009; Dolganiuc et al., 2003; Laliberte et al., 2006; Laliberte et al., 2007; Leser and Lamb, 2005; Manié et al., 2000; Robinzon et al., 2009; Scheiffele et al., 1999; Takeda et al., 2003; Vincent et al., 2000; Xiangjie and Whittaker, 2003). Proteins of several paramyxoviruses (non-segmented (−)ssRNA viruses) are also targeted to the raft domains of infected cells (e.g. Sendai virus, RSV, measles) (Ali and Nayak, 2000; Brown et al., 2002; Brown et al., 2004; Fleming et al., 2006; Manié et al., 2000; Marty et al., 2004; McDonald et al., 2004; Oomens et al., 2006). Few studies have reported an essential function of rafts during paramyxovirus infection. Rafts play a role during NDV (an avian paramyxovirus) infection (Dolganiuc et al., 2003; Laliberte et al., 2006; Laliberte et al., 2007) since it is required for release of infectious NDV particles. Budding of parainfluenza 5 virus requires caveolin, a component of raft-like structure known as caveolae (Ravid et al., 2010). In the current study we demonstrated that rafts play an important role during RSV infection. Specifically, raft is required for release of infectious progeny RSV particles. Our studies revealed that raft (and cholesterol) is not required for RSV budding from infected cells. However, the infectiousness of released raft (and cholesterol) deficient RSV particles is severely compromised.
RSV is a lung-tropic virus that causes severe respiratory diseases during infancy, childhood, old age. The high morbidity and mortality associated with RSV infection is due to its ability to cause respiratory diseases like pneumonia and bronchiolitis (Collins et al., 2007; Hall, 2001; Hippenstiel et al., 2006). Several studies have suggested that rafts may play an important role during RSV life-cycle – a) RSV proteins associate with plasma membrane rafts during assembly, b) raft associated proteins were observed in purified RSV virion particles, and c) filamentous virion particle formation requires intact raft structure (Brown et al., 2002; Brown et al., 2004; Fleming et al., 2006; Marty et al., 2004; McDonald et al., 2004; Oomens et al., 2006; Yeo et al., 2009). The cholesterol lowering drug lovastatin also diminished RSV infection of HEp2 cells (the human cervical carcinoma cell-line) (Gower and Graham, 2001). However, the direct role of cholesterol during RSV infection was not evaluated in these cells using a cholesterol-specific drug. Moreover, the role of cholesterol rich raft during RSV life-cycle was unknown. In the current study we have illustrated that rafts are required for release of infectious progeny RSV virion particle.
Lipid raft domains are localized on the exoplasmic side of the plasma membrane and are enriched with cholesterol and sphingolipids (Pike, 2003; Silvius, 2003). The raft domain forms a localized rigid (less fluid) environment due to the clustering of cholesterol molecules and the saturated fatty acyl chains of the sphingolipids. Various proteins (e.g. GPI-anchored CD59 protein, caveolin-1 etc) have high affinity for lipid rafts and therefore, they partition themselves in the raft domains. Such partitioning may occur during intracellular trafficking, whereby these proteins segregate in the lipid rafts of vesicles destined to deliver their cargo to the plasma membrane. Plasma membrane rafts have multiple functions during normal cellular processes. They play an important role during intracellular trafficking/targeting originating from the cell surface and are required for cell-to-cell adhesion. Apart from these functions, rafts act as a cell surface platform for initiating key signal transduction pathways (Simons and Toomre, 2000; Ning et al., 2006). The rigid (less fluid) micro-environment in the plasma membrane provided by the cholesterol and sphingolipids (key components of rafts) facilitates homotypic and heterotypic interaction between plasma membrane associated proteins/receptors. These interactions may only occur in the raft “micro-domains” since rigid (less fluid) environment provides limited flexibility to raft resident proteins which promotes their functional interactions.
Our study demonstrated that intact cell surface rafts are essential for release of progeny infectious RSV particles from infected cells. In context to enveloped viruses it is speculated that intracellular-trafficking/seclusion of viral membrane associated proteins (e.g. RSV envelope proteins F and G and matrix protein M associates with membrane) in the rafts results in utilization of rafts as the platform for assembly and budding. In addition, cellular proteins involved in viral release (Harty et al., 2001; Irie et al., 2004; Okumura et al., 2008; Usami et al., 2009) may also localize in rafts to facilitate assembly/budding of progeny virion particles. For example, Rho A (Gower et al., 2001; Gower et al., 2005; Pastey et al., 2000) and FIP2 (Rab11 family interacting protein 2) (Utley et al., 2008) have been shown to play an important role during RSV budding. Rho A activation by RSV is required for filamentous virus formation during morphogenesis (Gower et al., 2005). However, active Rho A is dispensable for RSV infection (Gower et al., 2005). Interestingly, both Rho A and FIP2 has been implicated in functioning via raft domain of plasma membrane (Chu et al., 2009; Lacalle et al., 2002). One could envision that raft disruption leads to RSV budding defect due to lack of functional scaffolding of host proteins like FIP2. However, it is a highly unlikely scenario, since virus budding was preserved following plasma membrane raft disruption (Fig. 4A) as deduced from the viral protein content of the purified virion particles. In contrary, our study suggested that release of infectious RSV particle was compromised following raft disruption. In that context, we also observed reduced virion-associated cholesterol levels in RSV particles released from raft disrupted cells (Fig. 4B, Table. 1). In the future, we will investigate the role of virion associated cholesterol (and rafts) in the RSV infection process.
In summary, our study has uncovered a critical role of rafts during RSV infection. Rafts are required for release of infectious RSV virion particles, since intact raft domains are necessary for “loading” cholesterol into the RSV virion particle.
Virus and cells
RSV (A2 strain) was propagated in CV-1 cells (Kota et al., 2008; Sabbah et al., 2009). VSV (Indiana serotype, Mudd–Summers strain) was propagated in BHK-21 cells (Basu et al., 2006; Bose et al., 2003). RSV and VSV were purified by centrifugation on discontinuous sucrose gradients as described previously (Ueba, 1978). Human lung epithelial A549 cells and normal primary human bronchial epithelial cells (NHBE cells were purchased from Lonza) were maintained in DMEM (supplemented with 10% fetal calf serum or FCS, penicillin, streptomycin, and glutamine) and Bronchial Epithelial Cell Basal Medium (containing BPE, Hydrocortisone, hEGF, Epinephrine, Transferrin, Insulin, Retinoic Acid, Triiodothyronine, GA-1000) (Clonetics/Lonza), respectively. Normal human embryonic lung (HEL) fibroblasts (ATCC) and Niemann-Pick syndrome type C human (NPC) fibroblasts (NIH-Coriell Cell Repository) were maintained in DMEM (supplemented with 10% FCS, non-essential amino acids, glutamine, vitamins). RSV titer was monitored by plaque assay analysis with CV-1 cells as described earlier (Kota et al., 2008; Sabbah et al., 2009).
Virus infection
To study the role of rafts during RSV infection, A549 and NHBE cells were treated with cholesterol disrupting drug methyl-beta-cyclodextrin (MBCD) (Sigma-Aldrich). Cells were treated with MBCD after virus adsorption stage. RSV (0.2 MOI) or VSV (0.1 MOI) was added to cells (A549 and NHBE) for 1.5h (adsorption stage). Following adsorption, the cells were washed and fresh medium containing 5 mM MBCD was added. After 1h MBCD incubation, cells were washed and once again fresh medium was added in the presence (for MBCD treated cells) or absence (control cells) of lovastatin (Sigma-Aldrich) (4 µg/ml). Medium supernatant was collected at either 24h (for A549) or 48h (for NHBE) post infection to assess viral titer by plaque assay.
HEL and NPC cells were also infected with RSV (0.5 MOI) or VSV (0.5 MOI). At 36h post-infection, medium supernatant was utilized to assess viral titer by plaque assay. In addition, light microscope was utilized to visualize cell morphology and cytopathic effect in RSV infected HEL and NPC cells.
Virus release assay
Virus release or budding assay was essentially performed as described previously (Bose et al., 2001). In order to investigate the effect of raft disruption (i.e. MBCD treatment) on RSV release, A549 cells were infected with RSV (1 MOI). At 8h post-infection, fresh medium containing MBCD (5mM) was added to washed cells and the cells were incubated with MBCD for 1h at 37°C. After 1h, cells were washed and fresh medium was added in the presence of lovastatin (4 µg/ml) and cycloheximide (30 µg/ml cycloheximide was added to accumulate an intracellular viral protein pool in the absence of de novo protein synthesis). Following 3h incubation, cells were washed and fresh medium (+/− lovastatin, but with no cycloheximide) was added to the cells to examine the assembly and release/budding of the accumulated viral proteins as virion particles in the presence of intact (MBCD and lovastatin untreated cells) and disrupted (MBCD and lovastatin treated cells) rafts. After 12 h the medium supernatants were assessed for viral titer by plaque assay.
Budding assay with NPC and HEL cells were performed similar to A549 cells but devoid of MBCD treatment. NPC and HEL cells were infected with RSV (1 MOI). At 10h post-infection, fresh medium containing cycloheximide (30 µg/ml cycloheximide was added to accumulate an intracellular viral protein pool in the absence of de novo protein synthesis) was added to washed cells and the cells were incubated with cycloheximide for 3h. Following 3h incubation, cells were washed and fresh medium (with no cycloheximide) was added to the cells to examine the assembly and release/budding of the accumulated viral proteins as virion particles. After 12 h the medium supernatants were assessed for viral titer by plaque assay.
Radioactive labeling
The viral protein composition of released progeny virus was examined by labeling cells with 35S-methionone. A549 cells were infected with RSV (2 MOI). At 8h post-infection, cells were washed and incubated with fresh medium containing 5mM MBCD. After 1h, MBCD medium was replaced with methionine free medium containing +/− lovastatin (4 µg/ml). Following 2h incubation with methionine free medium, cells were pulsed with 35S-methionone for 4h. The radioactive medium was replaced with medium containing cold methionine +/− lovastatin. After 12h, 35S-RSV was purified from medium supernatant and the radioactive virus was subjected to SDS-PAGE and autoradiography to visualize viral protein composition.
Immunofluorescence analysis
A549 cells grown on coverslips were either untreated or incubated with 5 mM MBCD for 1h. Fresh medium containing lovastatin (4 µg/ml) was added to washed cells. After 16h, cells were incubated with FITC conjugated cholera-toxin subunit-B (CHTX) (Sigma-Aldrich). Cells were fixed with 3.7% formaldehyde in PBS and visualized by confocal microscopy (Leica CISM confocal laser-scanning microscope).
Cholesterol estimation assay
Untreated and MBCD treated cells were lysed and total cholesterol content was measured by cholesterol quantitation kit (MBL Intl) according to the manufacturer’s specification. Virion cholesterol amount was also estimated in purified RSV virion particles (RSV purified from the medium supernatant) by using the cholesterol quantitation kit (MBL Intl).
Conclusions
Plasma membrane cholesterol-rich lipid rafts play a critical role during RSV infection, since raft domains are required for release of infectious progeny RSV virion particles.
Highlights
  • - 
    Plasma membrane cholesterol-rich lipid rafts are required for human respiratory syncytial virus (RSV) infection.
  • - 
    Rafts are essential for release of infectious RSV virion particles.
  • - 
    Rafts serve as a platform to load cholesterol into the envelope of RSV particles.
Supplementary Material
01
Acknowledgments
This work was supported by National Institutes of Health grant AI083387 (S.B.). A.S. and V.M. are supported by NIH/NIDCR grant # DE14318 for the COSTAR program. We thank Dr. Mark E. Peeples (The Ohio State University and Nationwide Children’s Hospital, Columbus, OH) for helpful suggestions and critically reading the manuscript. We also thank the Optical Imaging Core Facility (University of Texas Health Science Center at San Antonio) for confocal images.
Footnotes
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
  • Ali A, Nayak DP. Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein. Virology. 2000;276:289–303. [PubMed]
  • Allsopp RC, Lalo U, Evans RJ. Lipid raft association and cholesterol sensitivity of P2×1–4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2×1 receptors. J. Biol. Chem. 2010;285:32770–32777. [PMC free article] [PubMed]
  • Ayota E, Müller N, Klett M, Scneider-Schaulies S. Measles virus interacts with and alters signal transduction in T-cell lipid rafts. J. Virol. 2004;78:9552–9559. [PMC free article] [PubMed]
  • Basu M, Maitra RK, Xiang Y, Meng X, Banerjee AK, Bose S. Inhibition of vesicular stomatitis virus infection in epithelial cells by alpha interferon-induced soluble secreted proteins. J. Gen. Virol. 2006;87:2653–2662. [PubMed]
  • Bavari S, Bosio CM, Wiegand E, Ruthel G, Will AB, Geisbert TW, Hevey M, Schmaljohn C, Schmaljohn A, Aman MJ. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 2002;195:593–602. [PMC free article] [PubMed]
  • Bose S, Malur A, Banerjee AK. Polarity of human parainfluenza virus type 3 infection in polarized human lung epithelial A549 cells: Role of microfilament and microtubule. J. Virol. 2001;75:1984–1989. [PMC free article] [PubMed]
  • Bose S, Mathur M, Bates P, Joshi N, Banerjee AK. Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype. J. Gen. Virol. 2003;84:1687–1699. [PubMed]
  • Brown G, Rixon HW, Sugrue RJ. Respiratory syncytial virus assembly occurs in GM1-rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin-1. J. Gen. Virol. 2002;83:1841–1850. [PubMed]
  • Brown G, Jeffree CE, McDonald T, Rixon HW, Aitken JD, Sugrue RJ. Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection. Virology. 2004;327:175–185. [PubMed]
  • Chu B, Ge BL, Xie C, Zhao Y, Miao HH, Wang J, Li BL, Song BL. Requirement of myosin Vb.Rab11a.Rab11-FIP2 complex in cholesterol-regulated translocation of NPC1L1 to the cell surface. J. Biol. Chem. 2009;284:22481–22490. [PubMed]
  • Clemente R, de Parseval A, Perez M, de la Torre JC. Borna disease virus requires cholesterol in both cellular membrane and viral envelope for efficient cell entry. J. Virol. 2009;83:2655–2662. [PMC free article] [PubMed]
  • Collins PL, McIntosh K, Chanock RM. Respiratory syncytial virus. In: Knipe DM, Howley PM, editors. Fields Virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. pp. 1601–1640.
  • Dolganiuc V, McGinnes L, Luna EJ, Morrison TG. Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts. J. Virol. 2003;77:12968–12979. [PMC free article] [PubMed]
  • Fleming EH, Kolokoltsov AA, Davey RA, Nichols JE, Roberts NJ., Jr Respiratory syncytial virus F envelope protein associates with lipid rafts without a requirement for other virus proteins. J. Virol. 2006;80:12160–12170. [PMC free article] [PubMed]
  • Gower TL, Graham BS. Antiviral activity of lovastatin against respiratory syncytial virus in vivo and in vitro. Antimicrob. Agents Chemother. 2001;45:1231–1237. [PMC free article] [PubMed]
  • Gower TL, Peeples ME, Collins PL, Graham BS. RhoA is activated during respiratory syncytial virus infection. Virology. 2001;283:188–196. [PubMed]
  • Gower TL, Pastey MK, Peeples ME, Collins PL, McCurdy LH, Hart TK, Guth A, Johnson TR, Graham BS. RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology. J Virol. 2005;79:5326–5336. [PMC free article] [PubMed]
  • Hall CB. Respiratory syncytial virus and parainfluenza virus. N. Engl. J. Med. 2001;344:1917–1928. [PubMed]
  • Harder T, Scheiffele P, Verkade P, Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell. Biol. 1998;141:929–942. [PMC free article] [PubMed]
  • Harty RN, Brown ME, McGettigan JP, Wang G, Jayakar HR, Huibregtse JM, Whitt MA, Schnell MJ. Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction. J. Virol. 2001;75(22):10623–10629. [PMC free article] [PubMed]
  • Henderson LP, Lin L, Prasad A, Paul CA, Chang TY, Maue RA. Embryonic striatal neurons from Niemann-Pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness. J. Biol. Chem. 2000;275:20179–20187. [PubMed]
  • Hippenstiel S, Opitz B, Schmeck B, Suttorp N. Lung epithelium as a sentinel and effector system in pneumonia--molecular mechanisms of pathogen recognition and signal transduction. Respir. Res. 2006;7:97. [PMC free article] [PubMed]
  • Ikonen E, Holtta-Vuori M. Cellular pathology of Niemann-Pick type C disease. Semin. Cell Dev. Biol. 2004;15:445–454. [PubMed]
  • Irie T, Licata JM, McGettigan JP, Schnell MJ, Harty RN. Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A. J. Virol. 2004;78:2657–2665. [PMC free article] [PubMed]
  • Koike T, Ishida G, Taniguchi M, Higaki K, Ayaki Y, Saito M, Sakakihara Y, Iwamori M, Ohno K. Decreased membrane fluidity and unsaturated fatty acids in Niemann-Pick disease type C fibroblasts. Biochim. Biophys. Acta. 1998;1406:327–335. [PubMed]
  • Kota S, Sabbah A, Chang TH, Harnack R, Xiang Y, Meng Y, Bose S. Role of human beta-defensin-2 during tumor necrosis factor-α/NF-κB mediated innate anti-viral response against human respiratory syncytial virus. J. Biol. Chem. 2008;283:22417–22429. [PubMed]
  • Lacalle RA, Mira E, Gomez-Mouton C, Jimenez-Baranda S, Martinez-A C, Manes S. Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation. J. Cell Biol. 2002;157:277–289. [PMC free article] [PubMed]
  • Laliberte JP, McGinnes LW, Peeples ME, Morrison TG. Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles. J. Virol. 2006;80:10652–10662. [PMC free article] [PubMed]
  • Laliberte JP, McGinnes LW, Morrison TG. Incorporation of functional HN-F glycoprotein-containing complexes into Newcastle disease virus is dependent on cholesterol and membrane lipid raft integrity. J. Virol. 2007;81:10636–10648. [PMC free article] [PubMed]
  • Leser GP, Lamb RA. Influenza virus assembly and budding in raft-derived microdomains: a quantitative analysis of the surface distribution of HA, NA and M2 proteins. Virology. 2005;342:215–227. [PubMed]
  • Manié SN, de Breyne S, Vincent S, Gerlier D. Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly. J. Virol. 2000;74:305–311. [PMC free article] [PubMed]
  • Marty A, Meanger J, Mills J, Shields B, Ghildyal R. Association of matrix protein of respiratory syncytial virus with the host cell membrane of infected cells. Arch. Virol. 2004;149:199–210. [PubMed]
  • McDonald TP, Pitt AR, Brown G, Rixon HW, Sugrue RJ. Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis. Virology. 2004;330:147–157. [PubMed]
  • Medigeshi GR, Hirsch AJ, Streblow DN, Nikolich-Zugich J, Nelson JA. West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin. J. Virol. 2008;82:5212–5219. [PMC free article] [PubMed]
  • Ning Y, Buranda T, Hudson LG. Activated epidermal growth factor receptor induces integrin alpha2 internalization via caveolae/raft-dependent endocytic pathway. J. Biol. Chem. 2006;282:6380–6387. [PubMed]
  • Okumura A, Pitha PM, Harty RN. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc. Natl. Acad. Sci U.S.A. 2008;105:3974–3979. [PubMed]
  • Oomens AG, Bevis KP, Wertz GW. The cytoplasmic tail of the human respiratory syncytial virus F protein plays critical roles in cellular localization of the F protein and infectious progeny production. J. Virol. 2006;80:10465–10477. [PMC free article] [PubMed]
  • Pastey MK, Gower TL, Spearman PW, Crowe JE, Jr, Graham BS. A RhoA-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3. Nat. Med. 2000;6:35–40. [PubMed]
  • Pike LJ. Lipid rafts: bringing order to chaos. J. Lipid. Res. 2003;44:655–667. [PubMed]
  • Ravid D, Leser GP, Lamb RA. A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5. J Virol. 2010;84:9749–9759. [PMC free article] [PubMed]
  • Robinzon S, Dafa-Berger A, Dyer MD, Paeper B, Proll SC, Teal TH, Rom S, Fishman D, Rager-Zisman B, Katze MG. Impaired cholesterol biosynthesis in a neuronal cell line persistently infected with measles virus. J. Virol. 2009;83:5495–5504. [PMC free article] [PubMed]
  • Sabbah A, Chang TH, Harnack R, Frohlich V, Dube PH, Tominaga K, Xiang Y, Bose S. Activation of innate immune antiviral response by Nod2. Nature. Immunol. 2009;10:1073–1080. [PMC free article] [PubMed]
  • Scheiffele P, Rietveld A, Wilk T, Simons K. Influenza viruses select ordered lipid domains during budding from the plasma membrane. J. Biol. Chem. 1999;274:2038–2044. [PubMed]
  • Silvius JR. Role of cholesterol in lipid raft formation: lessons from lipid model systems. Biochim. Biophys. 2003;1610:174–183. [PubMed]
  • Simons K, Toomre D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 2000;1:31–39. [PubMed]
  • Takeda M, Leser GP, Russel CJ, Lamb RA. Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc. Natl. Acad. Sci. U.S.A. 2003;100:14610–14617. [PubMed]
  • Tang Y, Leao IC, Coleman EM, Broughton RS, Hildreth JE. Deficiency of niemann-pick type C-1 protein impairs release of human immunodeficiency virus type 1 and results in Gag accumulation in late endosomal/lysosomal compartments. J. Virol. 2009;83:7982–7995. [PMC free article] [PubMed]
  • Ueba O. Respiratory syncytial virus. I. Concentration and purification of the infectious virus. Acta Med Okayama. 1978;32:265–272. [PubMed]
  • Usami Y, Popov S, Popova E, Inoue M, Weissenhorn W, Göttlinger HG. The ESCRT pathway and HIV-1 budding. Biochem. Soc. Trans. 2009;37:181–184. [PubMed]
  • Utley TJ, Ducharme NA, Varthakavi V, Shepherd BE, Santangelo PJ, Lindquist ME, Goldenring JR, Crowe JE., Jr Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2. Proc. Natl. Acad. Sci. U.S.A. 2008;105:10209–10214. [PubMed]
  • Vincent S, Gerlier D, Manié SN. Measles virus assembly within membrane rafts. J. Virol. 2000;74:9911–9915. [PMC free article] [PubMed]
  • Xiangjie S, Whittaker GR. Role for Influenza virus envelope cholesterol in virus entry and infection. J. Virol. 2003;77:12543–12551. [PMC free article] [PubMed]
  • Xu S, Huo J, Gunawan M, Su IH, Lam KP. Activated dectin-1 localizes to lipid raft microdomains for signaling and activation of phagocytosis and cytokine production in dendritic cells. J. Biol. Chem. 2009;284:22005–22011. [PubMed]
  • Yeo DS, Chan R, Brown G, Ying L, Sutejo R, Aitken J, Tan BH, Wenk MR, Sugrue RJ. Evidence that selective changes in the lipid composition of raft-membranes occur during respiratory syncytial virus infection. Virology. 2009;386:168–182. [PubMed]