Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses

Abstract

Cholesterol-rich lipid raft microdomains in the plasma membrane are considered to play a major role in the enveloped virus lifecycle. However, the functional role of cholesterol in assembly, infectivity and stability of respiratory RNA viruses is not fully understood. We previously reported that depletion of cellular cholesterol by cholesterol-reducing agents decreased production of human parainfluenza virus type 1 (hPIV1) particles by inhibiting virus assembly. In this study, we analyzed the role of cholesterol on influenza A virus (IAV) and respiratory syncytial virus (RSV) production. Unlike hPIV1, treatment of human airway cells with the agents did not decrease virus particle production. However, the released virions were less homogeneous in density and unstable. Addition of exogenous cholesterol to the released virions restored virus stability and infectivity. Collectively, these data indicate a critical role of cholesterol in maintaining IAV and RSV membrane structure that is essential for sustaining viral stability and infectivity.

Keywords: Antiviral therapy; Cholesterol; Lipid rafts; Respiratory viruses; Virus stability.

Figure 1. Cholesterol is required for infectious IAV and RSV production

Cells untreated or treated with the drugs for 24 h were infected with H1N1 (A, left), H3N2 IAV (A, right) or RSV (B, right) at MOI of 3, and cultured in presence of drugs for an additional 24 h. Harvested viruses were saved in −80°C freezer, and virus titers were measured by TCID₅₀ or IF assays (n=3). (B, left) Cells either untreated or treated with the drugs were infected with RSV at MOI of 0.1 and cultured in presence or absence of the drugs for an additional 24 h and 48 h. Infected cells were detected by IF analysis using anti-NP mAb (n=3). **; P<0.01, *; P<0.05 (Student t-test).

Figure 2. Cholesterol depletion does not prevent IAV or RSV entry

Cells untreated or treated with the drugs were infected with viruses at MOI of 0.5 and cultured for 24 h. Cells were fixed and viral NP was detected by IF assay using specific antibodies. The NP positive cells were counted and presented as bar graphs below (n=3).

Figure 3. Cholesterol depletion does not affect IAV or RSV protein synthesis

Cells untreated or treated with the drugs were infected with IAV (H1N1) (A) or RSV (B) at MOI of 3 and cultured in the presence of drugs for 24 h. HA, NP and M1 of IAV, and G and N of RSV in the cell lysates were detected by Western blot analysis using specific antibodies. Cellular actin was detected as a loading control.

Figure 4. Cholesterol depletion does not decrease surface accumulation of IAV and RSV proteins

Cells untreated or treated with the drugs were infected with hPIV1, IAV (H1N1) or RSV at MOI of 3 and cultured in the presence of drugs for 24 h. (A) Cells were fixed and surface viral proteins expression was determined by IF assay using specific antibodies. (B) Quantities of viral glycoproteins were determined by cell surface ELISA. **; P<0.01 (Student t-test).

Figure 5. Characterization of viruses released from cholesterol depleted cells

(A) Distribution of released virions in 10–40% sucrose gradient was determined by Western blot analysis using HA (left) or NP (right) Abs. Percentages of proteins in each fraction are shown as a line graph. (B) Released virions were purified through 5–40% sucrose step gradient ultracentrifugation. Protein and cholesterol contents in the purified virions were quantitated (n=3). *; P<0.05 (Student t-test).

Figure 6. Cholesterol depletion affects virus stability

Cells untreated or treated with the drugs were infected with IAV (H1N1) (left panel) or RSV (right panel) at MOI of 3 and cultured in the presence of drugs for 24 h. Released virions were titrated either fresh or after incubating at RT for 24 h, 48 h or at −80°C for 24 h (1 x thaw). Titers of the released virions are shown as bar graphs (n=3). **; P<0.01, *; P<0.05 (Student t-test).

Figure 7. Cholesterol is required for maintaining virus stability and infectivity

Cells untreated or treated with the drugs were infected with IAV (H1N1) at MOI of 3 and cultured in the presence of drugs for 24 h. Released virions were kept at RT with varying concentration of cholesterol for 48 h. Virus titer were then measured by TCID₅₀ assay (n=3). *; P<0.05 (Student t-test).