| Bovine herpesvirus-1(BoHV-1) is an important pathogen of cattle, which mainly causes severe respiratory disease. Also it is responsible for several other infections, such us conjunctivitis, genital lesion, abortion, enteritis and encephalitis. Inflammation and cell necrosis and death of respiratory epithelia, and immunosuppression caused by the virus infection often predispose cattle to secondary or opportunistic infections in particular Mannheimia haemolytica and Haemophilus somnus, which results in enhanced morbidity and mortality. Abortions caused by the virus infection of the genital tract results in significant economic loss in the cow industry worldwide. Infection with herpesviruses is commonly characterized by the ability to establish latent infection, mainly in ganglion cells and tonsils. Once infected, the virus persists for life and may be re-activated at intervals if the host is subjected to stress or immunosuppression, for example by dexamethasone treatment. The continuous circulation of the virus among the bovine population is the cause of serious economic losses for the cattle industry worldwide.For the virus successful infection of susceptible cells, series of cellular molecules must be involved for the facility of a complete life cycle. And during the cause of the virus-cell interactions, some cellular signaling pathways were involved. Thus, it is of great significance to investigate the virus infection-associated cellular molecules andsingnaling pathways, for the dissecting of virus replication mechanism. Above basic research on virus-cell interaction could also contribute to the pharmacology development, for providing some target molecule for the novel medicine design. Lipid rafts, specific membrane microdomains enriched in cholesterol,(glyco)sphingolipids and phospholipids, which tightly packaged and ordered, are known to be involved in the regulation of various biological phenomena, including membrane transport protein sorting during endocytosis and exocytosis, cell adhesion, migration, cell apoptosis, synaptic transmission and organization of the cytoskeleton et al. Accumulating evidence suggests that many pathogens require Lipid rafts at multiple stages of their life cycles. However, as an important component of Lipid raft, the role of cholesterol in the life cycle of BoHV-1infection of Madin-Darby bovine kidney (MDBK) cells remains unknown, thus it deserves extensive investigation.Cholesterol is an important component of membrane Lipid raft, by the surveys of its role for the virus infection will contribute to understand the function of Lipid raft in the virus infection. Methyl-β-cyclodextrin (MβCD) is an effective reagent to sequester cell membrane cholesterol and was used for this research. Here, we examined the role of cholesterol for both viral envelope and target cell membrane for BoHV-1infection. Cholesterol depletion by pretreatment of MDBK cells with MβCD, inhibited the production of BoHV-1in a dose-dependent manner. This inhibitory effect was partially reversed by cholesterol replenishment, indicating that the reduction was caused by cholesterol depletion. Cholesterol depletion at the stage only had a mild effect on the virus production. However, cell membrane cholesterol depletion did not reduce the virus attachment. In addition, treatment of BoHV-1particles with MβCD also reduced the virus infectivity significantly and the effect was partially reversed by addition of exogenous cholesterol. Taken together, these data implicated that cell membrane cholesterol mainly contributed to BoHV-1entry into MDBK cells and the viral envelope cholesterol was also essential for the virus infectivity. Why Lipid raft is associated with the virus entry process is not known. We hypothesis that some molecules in the Lipid raft is associated with some cellular signal transductions as well some process of the virus entry. However, as to the information of cellular signaling pathways involved in BoHV-1infectin of into MDBK cells is not known. Phosphatidylinositol3-kinases (PI3K) PI3K/Akt is an important signaling pathway which executes series of biological function. A number of molecules are directly or indirectly regulated by Akt, to carry out PI3K-regulated responses such as cell survival, growth, proliferation, angiogenesis, metabolism, and migration. The members of mitogen-activated protein kinase (MAPK) superfamily respond to diverse cellular stimuli through transducing signals from the cell membrane to the nucleus. Extracellular signal-regulated kinases1and2(Erkl/2) is a member of MAPK family, which regulate a wide range of cellular functions including cell proliferation, transformation, differentiation, cell survival and death. So far it is not clear whether PI3K/Akt and MAPK/Erkl/2signaling pathways are involved in the process of BoHV-linfection of MDBK cells.This study indicated that infection of MDBK cells with BoHV-1induced an early-stage transient and a late-stage sustained activation of both PI3K/Akt and MAPK/Erkl/2signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective and potent inhibitors Ly294002for PI3K and U0126for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002caused a1.5-log reduction in virus titer, while U0126had little effect on the virus production. And the inhibition effect mainly occurred at the post-entry stage of the virus replication cycle. This was the first time to reveal that BoHV-1actively induced both PI3K/Akt and MAPK/Erkl/2signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.In previous study, we reported that cholesterol-rich Lipid raft is involved in the life cycle of BoHV-1infection of MDBK cells, especially for the virus entry process. During the virus binding and/or entry process, both PI3K/Akt and MAPK/Erkl/2signaling pathways are activated. But whether Lipid raft was involved in the virus induced signaling stimulation process was not clear. The following experiments revealed that cholesterol depletion had a minor influence on the virus triggered signaling pathways. Taken together, here for the first time experimental data revealed that cholesterol was required for the virus effective infection of MDBK cells at the entry stage, and it induced an early-stage transient and a late-stage sustained activation of both PI3K/Akt and MAPK/Erkl/2signaling pathway. The activation of PI3K/Akt signaling is of great significance for the virus replication especially at the post-entry stage. In addition, the depletion of cellular cholesterol had minor effect on the virus induced PI3K/Akt and MAPK/Erkl/2signaling pathways activation. |
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