Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade

Abstract

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MβCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

Keywords: Nipah virus; cholesterol; entry; fusion; henipavirus; membrane; paramyxovirus.

FIG 1

Depleting cellular cholesterol reduces cell-cell fusion. (A) The total cellular cholesterol concentration was measured with an Amplex red cholesterol kit after treatment with MβCD and 10 μM lovastatin. Cholesterol concentrations were normalized to those in mock-treated cells. (B) NiV F/G-transfected HEK293T cells were treated with increasing concentrations of MβCD and 10 μM lovastatin. Cytotoxic positive-control cells were treated with H₂O₂ at 0.3%. Viability was quantified with CCK8 that measures dehydrogenase activity in live cells. Cholesterol levels and cell viability were measured 9 to 12 h after treatment. (C) The levels of cell-cell fusion were quantified by counting the syncytia formed. The minimum numbers of nuclei inside syncytia were counted, considering a syncytium as having 4 or more nuclei within a common cell membrane. Nuclei in syncytia per random ×200 field were normalized to the no-treatment mock control, set at 100%. (D) Representative fields of syncytia, after treatment with 10 mM MβCD and 10 μM lovastatin, circled in red. (E) The levels of CSE of NiV F after cholesterol depletion were measured using polyclonal rabbit antibody 835 against NiV F (39, 40). G was detected using a monoclonal anti-HA phycoerythrin (PE) antibody. After the removal of membrane cholesterol, the levels of ephrinB2 binding to NiV G were measured using soluble ephrinB2 fused to human Fc. (F) Representative Western blot analysis of NiV F expression and cleavage of mock-treated and cholesterol-depleted cells. Data shown are averages from three independent experiments ± standard deviations (SD). Statistical significance was determined with a one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 2

Enriching cellular cholesterol increases cell-cell fusion. (A) The total cellular cholesterol concentration was measured with an Amplex red cholesterol kit after treatment with increasing concentrations of a cholesterol/MβCD (1:20) solution. Cholesterol concentrations were normalized to those in mock-treated cells. (B) NiV F/G-transfected HEK293T cells were treated with increasing concentrations of cholesterol/MβCD. Cytotoxic positive-control cells were treated with H₂O₂ at 0.3%. Viability was quantified with CCK8 that measures dehydrogenase activity in live cells. Cholesterol levels and cell viability were measured 9 to 12 h after treatment. (C) The levels of cell-cell fusion were quantified by counting syncytia. The minimum number of nuclei necessary to be considered a syncytium was 4 or more within a common cell membrane. Nuclei inside syncytia per random ×200 field were normalized to the no-treatment mock control, set at 100%. (D) Representative fields of syncytia, after treatment with 5 mM cholesterol/MβCD, circled in red. (E) The levels of CSE of NiV F after cholesterol enrichment were measured using polyclonal rabbit antibody 835 against NiV F. G was quantified using a monoclonal anti-HA PE antibody. After intercalation of membrane cholesterol, the levels of ephrinB2 binding to NiV G were measured with ephrinB2 fused to human Fc. (F) Representative Western blot analysis of NiV F expression and cleavage after cholesterol enrichment. Data shown are averages from three independent experiments ± SD. Statistical significance was determined with a one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Modifying the concentration of membrane cholesterol alters viral particle infectivity. (A) The cholesterol concentration in NiV/VSV-pseudotyped virions, after treatment, was measured with an Amplex red cholesterol kit. Measurements were normalized to the mock-treated virus (set to 100%). (B) Western blot of NiV/VSV-pseudotyped virus from mock, cholesterol-depleted, and enriched NiV/VSV probed for NiV F/G glycoproteins. (C) Densitometry of NiV/VSV glycoproteins normalized to mock-treated NiV/VSV. PC, bald NiV/VSV produced with the pcDNA3.1(+) empty vector. Data shown are averages from three replicates ± SD. (D) Entry into HEK293T cells of NiV/VSV virions whose levels of cholesterol had been modified was measured with a Renilla luciferase kit (Promega). The RLU were quantified at 18 to 24 h postinfection. Data shown are from one representative of three independent experiments ± standard errors of the means (SEM). (E) Entry of NiV/VSV-pseudotyped virus into HEK293T target cells whose cholesterol levels had been modified. The data shown are averages from 4 independent experiments ± SD. Statistical significance was determined with a one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

The concentration of membrane cholesterol affects the levels of NiV F triggering. The degree of NiV F triggering in cells with altered levels of membrane cholesterol was determined by the use of a NiV F HR2-Cy5 peptide. The peptide binds to the exposed HR1 during the PHI conformation after the transition from 4°C to 37°C. Averages from three independent experiments ± SD are shown. Statistical significance was determined with a one-sample t test. *, P < 0.05; **, P < 0.01. (B) Representative examples of a pulldown of NiV G (HA tagged) and coimmunoprecipitation (Co-IP) of F (Au1 tagged) in transfected HEK293T cells. (C) Avidity was quantified by Western blot densitometry. The levels were calculated as (IP F)/(CL F × IP G) (40, 46). CL, cell lysates. The averages from four replicates with standard errors are shown.

FIG 5

Heterologous cell-cell fusion upon cholesterol alteration. (A to C) PK13 cells were transfected with either pcDNA3.1(+) or NiV F/G expression plasmids. Vero cells were dyed with CellTracker green (top right), DiI (bottom right), and Hoechst stain (bottom left) (merged filters, along with bright-field images, are in the top left panel) to visualize their cytoplasm, plasma membrane, and nuclei, respectively, before being overlaid onto a monolayer of PK13 cells for 4 h before fixation with 0.5% paraformaldehyde. Examples of no fusion (A), hemifusion (B), and full fusion (C) are shown. (D) Hemifusion events (percent) from a 6-well plate were completely surveyed at a magnification of ×40 to determine relative levels of hemifusion and full-fusion events for each treatment. Data shown represent averaged ratios of hemifusion/full cell-cell fusion with standard errors. (E) Average hemifusion and full-fusion counts per well with standard errors (n = 5).

FIG 6

Modifying the concentration of membrane cholesterol alters fusion pore formation. The effector cells expressing NiV F/G and DSP1 were overlaid, after treatment, onto the target cells expressing DSP2. The degree of fusion pore formation, reported with the EnduRen live-cell substrate (Promega), was quantified at 8 to 12 h postoverlay in cholesterol-depleted (A) or cholesterol-enriched (B) cells. Data shown are averages from 3 independent experiments ± SD. Statistical significance was determined with a one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Summary table and model of the roles of membrane cholesterol in NiV membrane fusion. (A) The CSE of NiV F and G, the ability of NiV G to bind ephrinB2, and F/G binding avidity were not affected when membrane cholesterol was altered. However, the levels of F triggering were altered. An increase in membrane cholesterol reduced F triggering, while a reduction increased F triggering. However, the levels of hemifusion did not change with a change in the membrane cholesterol concentration. Nevertheless, a late step in NiV membrane fusion, fusion pore formation, was significantly altered. WT, wild type. (B) Model for the role of membrane cholesterol in NiV membrane fusion. NiV membrane fusion begins with the binding of G to ephrinB2, and this interaction induces conformation changes within G, which ultimately activates a conformational cascade in F. During F’s transition from the PF to the PHI conformations, cholesterol-depleted cells (yellow arrow) had an increase in F triggering, while cholesterol-enriched cells (green arrow) had a reduction in F triggering, compared to mock-treated cells. Next, F merges the outer leaflets of the effector and target membranes. In cells with modified levels of cholesterol, the levels of quantified hemifusion events were not altered compared to mock-treated cells. NiV membrane fusion proceeds to fusion pore formation and expansion, which ultimately leads to viral entry or syncytium formation. Cholesterol-depleted cells yielded reduced levels, while cholesterol-enriched cells yielded high levels, of fusion pore formation compared to the mock cells. Overall, the cholesterol-depleted cells had reduced levels of syncytium formation, and the cholesterol-enriched cells had higher levels of cell-cell fusion. The sizes of the arrows indicate the relative levels of the phenotypes that they mark. Purple dots within the arrows represent the relative levels of cholesterol in those scenarios.