PRV infection upregulates Rab6 expression.
To determine the role of Rab6 in PRV infection, we evaluated the expression of Rab6 following a PRV challenge in vitro. Cells were infected with PRV-QXX for durations ranging from 0 to 24 hours, and both the mRNA and protein levels of Rab6 in the cells were measured. PRV infection resulted in increased Rab6 mRNA expression in both PK-15 and porcine alveolar macrophage (PAM) cells (Figs. 1A and B). Consistent with the mRNA levels, Rab6 protein levels in PK-15 and PAM cells were also elevated following PRV infection (Figs. 1C and D). Next, we investigated whether PRV upregulated the expression of Rab6 in vivo. Mice were either mock-infected or intranasally infected with PRV-QXX for three days. The lungs and spleens were assessed for Rab6 mRNA levels by quantitative real-time polymerase chain reaction (qRT-PCR), and the lungs were assessed for Rab6 protein levels by immunoblotting analysis. PRV infection resulted in elevated Rab6 expression in the tissues of infected mice compared with those of mock-infected mice (Figs. 1E–G). These results suggest that PRV promotes the expression of Rab6 in vitro and in vivo.
Rab6 overexpression promotes PRV infection.
To examine the role of Rab6 in PRV proliferation, we constructed a plasmid encoding FLAG-tagged Rab6. PK-15 cells were transfected with different concentrations of the FLAG-Rab6 plasmid or an empty vector for 24 hours, followed by infection with PRV-GFP. Fluorescent microscopy and fluorescence intensity analysis revealed a significant enhancement of the GFP fluorescence signal after overexpression of FLAG-Rab6, suggesting that Rab6 overexpression promoted PRV-GFP infection (Figs. 2A and B). We also examined the effect of Rab6 overexpression on PRV gB expression. Immunoblotting analysis showed a significant increase in PRV gB expression due to overexpression of FLAG-Rab6 (Fig. 2C). The plaque assay indicated that overexpression of Rab6 enhanced the production of viable progeny (Fig. 2D). Additionally, we constructed the GTP-bound mutant Rab6 Q72L (constitutively active) and the GDP-bound mutant Rab6 T27N (dominant negative). PK-15 cells were transfected with vector, FLAG-Rab6 Q72L and FLAG-Rab6 T27N. The overexpression of Rab6 Q72L was found to significantly elevate the expression of PRV gB, whereas the overexpression of Rab6 T27N had no discernible effect on the expression of PRV gB, as demonstrated by Western blot analysis (Fig. 2E). In the plaque assay, it was observed that overexpression of Rab6 Q72L resulted in enhanced virus proliferation, while Rab6 T27N did not affect virus proliferation (Fig. 2F). These results suggest that the overexpression of Rab6 promotes PRV infection.
Rab6 knockdown inhibits PRV infection.
To further validate the role of Rab6 in PRV infection, we depleted Rab6 using short hairpin RNA (shRNA)-mediated RNA interference. Both qRT-PCR and immunoblotting analyses showed that these three Rab6-targeting shRNAs exhibited significant knockdown efficiency in PK-15 cells (Figs. 3A and B). In addition, the knockdown of Rab6 did not affect cell viability (Fig. 3C). Next, we infected shcontrol, shRab6-1, shRab6-2, and shRab6-3 PK-15 cells with PRV-GFP and assessed viral proliferation using fluorescence microscopy. The observed decrease in fluorescence intensity in cells with Rab6 knockdown, in comparison to control cells, suggests an inhibitory effect of Rab6 knockdown on PRV-GFP replication (Figs. 3D and E). To further assess viral replication, PRV gB protein levels were analyzed by immunoblotting, showing notably lower levels in Rab6-knockdown cells than in control cells (Fig. 3F). Additionally, when cells were infected with PRV-QXX, the viral titer was found to be lower in the shRab6-1, shRab6-2, and shRab6-3 cells compared to the shcontrol cells (Fig. 3G). Taken together, the data showed that Rab6 knockdown inhibited the proliferation of PRV.
Rab6 is involved in PRV assembly
To gain a more thorough understanding, we investigated the precise stages at which Rab6 affects PRV's life cycle. Viral attachment to host cells is the first step in a viral infection. Initially, we incubated shcontrol and shRab6-1 cells with PRV at 4°C for 2 hours. After extensive washing three times with ice-cold phosphate-buffered saline (PBS), viral attachment was detected by qRT-PCR analysis of viral genome copy numbers on the cells. Additionally, the cells were then shifted to 37°C for 10 minutes to allow entry. After washing with 1 mg/mL trypsin to remove residual virions on the plasma membrane (PM), viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. Our results demonstrated that the knockdown of Rab6 had no effect on PRV attachment and entry (Figs. 4A and B). We delved deeper into viral assembly. Following the knockdown of Rab6, virus assembly efficiency decreased, as indicated by the viral assembly results (Fig. 4C). Moreover, We further analyzed the infectivity of intracellular and extracellular PRV progeny virus by a TCID50 assay. Both intracellular and extracellular PRV titers were decreased at 2 h postinfection (Figs. 4D and E). To further ascertain this, we transfected PK-15 cells with vector, FLAG-Rab6, FLAG-Rab6 CA, FLAG-Rab6 DN, and an empty vector plasmid; Upon infecting the cells with the virus, the efficiency of virus assembly was determined. The results demonstrate that the overexpression of FLAG-Rab6 significantly boosts the assembly of PRV compared to transfection with an empty vector (Fig. 4F). Simultaneously, it was observed that overexpressing FLAG-Rab6 CA increased PRV virus assembly compared to the overexpression of FLAG-Rab6, while overexpressing FLAG-Rab6 DN showed no impact on PRV assembly compared to the empty vector group (Fig. 4F). In conclusion, Rab6 is involved in PRV assembly.
Rab6 interacts with PRV gB and gE.
Evidence has been presented that Rab6 can impact the assembly stages of the virus. It is commonly understood that the glycoprotein of the herpes virus is a critical element in the process of assembling and releasing progeny viruses [31]. The virus's glycoprotein is encapsulated in the secondary envelope of vesicles derived from the trans-Golgi network, particularly during the assembly process of progeny viruses [32]. Initially, we conducted an analysis of the co-occurrence of PRV gB and gE in relation to Rab6. Our research demonstrated that when HA-tagged plasmids encoding gB and gE were expressed in PK-15 cells, the two proteins colocalized with Rab6 (Figs. 5A and B). Additionally, we transfected gI-HA, gM-HA, and gL-HA plasmids, and immunofluorescence detection showed that gI, gM, and gL did not colocalize with Rab6 (Figs. 5C-E). To validate the interaction between PRV gB and gE with Rab6, a co-immunoprecipitation (Co-IP) assay was performed on PK-15 cells that had been either mock-infected or infected with PRV. It was observed that PRV gB and PRV gE co-immunoprecipitated with Rab6, as illustrated in Figs. 5F and G. It appears that Rab6 is involved in the process of virus assembly through its interaction with PRV gB and gE.