Gossypol Affects Viral Replication by Inhibiting Pseudorabies Virus Adsorption

Figure 6

Gossypol exerted an antiviral effect by inhibiting PRV adsorption on the cell surface. (a) Antiviral effects of gossypol in PRV adsorption. PK-15 cells were precooled for 1 hr at 4°C, then incubated at 4°C for 1 hr with PRV HN1201 (MOI = 0.1 or MOI = 1) that had been precooled at 4°C, accompanied by different concentrations of gossypol (0, 0.1, 0.3, 1, or 3 µM). After the adsorption solution was discarded, the cells were washed three times with PBS and then cultured in maintenance solution at 37°C for 24 hr. The cells were frozen and thawed three times to collect the virus, and its TCID₅₀ was measured. , , and . (b) Antiviral effects of gossypol in PRV entry. PRV HN1201 (MOI = 0.1 or MOI = 1) was adsorbed onto PK-15 cells for 1 hr at 4°C. After the adsorption solution was discarded, the cells were washed three times with precooled PBS, and incubated at 37°C for 2 hr in maintenance solution containing the indicated concentration of gossypol (0.1, 0.3, 1, or 3 µM) or DMSO. The cells were then washed three times with PBS at room temperature, and further cultured in maintenance solution for 24 hr. The cells were frozen and thawed three times to collect the virus, and its TCID₅₀ was measured. (c) Antiviral effects of gossypol in PRV replication. PRV HN1201 (MOI = 1) was adsorbed onto PK-15 cells for 1 hr at 4°C. The cells were washed three times with PBS at room temperature and then cultured at 37°C for 2 hr in maintenance solution. The maintenance solution was replaced with fresh maintenance solution containing DMSO or 3 µM gossypol, and samples were collected at 4, 6, 8, 10, and 12 hr after the addition of gossypol. The TCID₅₀ was measured after the cells were frozen and thawed three times. (d) Antiviral effects of gossypol in PRV release. PRV HN1201 (MOI = 1) was adsorbed onto PK-15 cells for 1 hr at 4°C. The cells were then cultured at 37°C in maintenance solution for 12 hr, washed three times with PBS, and cultured in maintenance solution containing the indicated concentrations of gossypol (0.1, 0.3, 1, or 3 µM) or DMSO. The supernatants and cells were collected at different lengths of time (1, 2, 3, 4, 5, or 6 hr) after the addition of gossypol, and the TCID₅₀ of the virus was measured after the cells were frozen and thawed three times. (e, f) Inhibitory effect of different concentrations of gossypol during adsorption of PRV virus. PK-15 cells were inoculated with recombinant virus rPRV HN1201-EGFP-Luc (MOI = 0.01) in the presence of the indicated concentration of gossypol (0, 0.1, 0.3, 1, or 3 µM) at 4°C for 1 hr to allow adsorption. At 24 hr after inoculation, the effect of gossypol on the PRV adsorption was determined by fluorescence microscopy (e) and firefly luciferase activity assay (f) . (g, h) Detection of virus particle uptake on cell surfaces by atomic force microscopy. PRV HN1201 (MOI = 1,000) was adsorbed onto PK-15 cells in the presence of DMSO or 3 µM gossypol for 1 hr at 4°C. DMSO-treated noninoculated cells were included as a control. After the cells were washed three times with precooled PBS, the cell slides were removed and dried, then fixed face up on a glass slide, and the absorption of virus particles on the cell surface was detected under an atomic force microscope (g). The statistics of PRV particles in multiple fields of view was conducted (h) . (i) Virus attachment in the presence of different concentrations of gossypol (0, 0.1, 0.3, 1, or 3 µM). PRV HN1201 (MOI = 10) was adsorbed onto PK-15 cells in the absence or presence of gossypol (0, 0.1, 0.3, 1, or 3 µM) for 1 hr at 4°C. After the cells were washed three times with ice-cold PBS, the total DNA was extracted, PRV gH DNA was detected by qPCR, and virus attachment was detected , and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)