Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels

Abstract

Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

Figure 1

Effects of glutamine on viability of PK- 15 cells. PK-15 cells without (A) or with (B) PCV2 infection were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined by MTT assay. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).

Figure 2

Effects of different concentrations of glutamine on PCV2 replication. PK-15 cells were infected with PCV2 at an MOI of 1 and incubated in the complete medium for 24 h. Subsequently, PK-15 cells were grown in medium containing various concentrations of glutamine. After 48 h, the PK-15 cells were harvested to determine the number of PCV2 log ₁₀ DNA copies per 10⁶ cells (A) and the relative proportion of PCV2-infected cells (B and C) by real-time PCR and immunofluorescence microscopy, respectively. Values shown are means ± SD mean from three independent experiments. Asterisks indicate groups statistically significantly different from control by a 1-way ANOVA followed by least-significant difference test (**P < 0.01).

Figure 3

Glutamine supplementation blocks the promotion of PCV2 replication by glutamine starvation. PK-15 cells were infected with PCV2 at an MOI of 1 in complete medium for 24 h. These PK-15 cells were subsequently grown in medium containing various concentrations of glutamine for 48 h before the media of all treatment groups were changed to fresh media with 4 mM glutamine. The cells were incubated for another 48 h and then harvested to determine the number of PCV2 log ₁₀ DNA copies per 10⁶ cells (A) and the relative proportion of PCV2 infected cells (B and C). Groups were compared by a 1-way ANOVA followed by a least-significant difference test. Significant changes are indicated by *(P < 0.05) in comparison with control.

Figure 4

Effect of glutamine starvation on GSH levels, MDA concentration and PCV2 replication. After 24 h of PCV2 infection in complete medium, fresh glutamine-free medium with various concentrations of glutamine and BSO was added to replace the original medium. The cells were cultured for another 48 h before the determining of GSH levels (A and C), MDA concentration (B), the number of PCV2 log ₁₀ DNA copies per 10⁶ cells (D), and the proportion of PCV2 infected cells (E and F). Values are shown as mean ± SD from three independent experiments. Asterisks indicate groups statistically significantly different from control by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).

Figure 5

Glutamine starvation increases the level of p38 MAPK phosphorylation. After 24 h of PCV2 infection, the complete medium was removed, and fresh medium with various concentrations of glutamine was added for an additional 48 h of incubation. The protein levels of p38 phosphorylation were measured by Western blot. Values are shown as mean ± SD from three independent experiments. Asterisks indicate groups statistically significantly different from control by a 1-way ANOVA followed by a least-significant difference test (**P < 0.01).

Figure 6

Glutamine starvation increases the phosphorylation of p38 MAPK via the reduced GSH synthesis. PK-15 cells lysates were extracted to measure the protein levels of p38 phosphorylation by Western blot after incubation for 72 h in the absence of glutamine or in the presence of glutamine and BSO (A and B). Values are shown as mean ± SD from three independent experiments. Asterisks indicate groups statistically significantly different from control by a 1-way ANOVA followed by least-significant difference test (**P < 0.01).

Figure 7

Knockdown of p38 MAPK decreases PCV2 replication but does not affect the level of GSH and MDA in PK-15 cells. PCV2-infected cells were transfected with p38-specific or control siRNA. After 5 h of transfection treatment, the medium was removed, and fresh basal medium was added. After transfected-cells were cultured for another 72 h, the relative mRNA levels of p38 (A), the relative proteins levels of p38 and p-p38 (B and C), the number of PCV2 log ₁₀ DNA copies per 10⁶ cells (D), the relative proportion of infected cells (E), the level of GSH (F) and MDA (G) were assayed as described in Methods. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by least-significant difference test. Significant changes are indicated by **(P < 0.01) and ## (P < 0.01) in comparison with controls.

Figure 8

PCV2 replication promoted by glutamine starvation could be blocked by p38 knockdown in PK-15 cells. PCV2-infected cells were transfected with p38-specific, control or non siRNA. After 5 h of transfection treatment, the medium was removed, and fresh basal medium with various concentrations of glutamine was added. After transfected-cells were cultured for another 72 h, the number of PCV2 log ₁₀ DNA copies per 10⁶ cells (A) and the relative proportion of infected cells (B) were assayed by real-time PCR and immunofluorescence microscopy, respectively. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by least-significant difference test. Significant changes are indicated by **(P < 0.01) and # (P < 0.05) in comparison with controls.