Over the course of evolution, viruses have developed various strategies that modulate a variety of host cell signaling pathways to establish a moderate environment favourable for their survival or infection. In particular, delaying or evading cell apoptosis affords great advantages to the virus, facilitating virus replication, the spreading of progeny virus to neighboring cells and providing the protection for the progeny virus against cellular enzymes . PI3K/Akt and MAPK/Erk1/2 are such pathways that have attracted much interest due to its central role in the regulation of cell death, proliferation and survival.
In the present data we showed that the virus infection induced biphasic activation of PI3K/Akt and MAPK/Erk1/2 pathways in MDBK cells. Once exposed to BoHV-1, Akt and Erk1/2 were transiently phosphorylated, disappearing within a short period; after several hours another tier of persistent activation was observed (Figure 1a and Figure 2a). To preclude the possibility that cellular components in the virus stocks could activate the signaling pathways, the virus was pelleted by ultracentrifugation at 20,000 rpm (Beckman SW28 rotor) for 1 h as described elsewhere . The pelleted virus could enhance the level of both phosphoralated Akt and Erk1/2, however, the supernatant could not (data not shown). Take together the data demonstrate that BoHV-1 could active Akt and Erk1/2 in the infection of MDBK cells. In addition, we showed that inhibition of PI3K resulted in a significant reduction of the progeny virus production.
Further investigation revealed that sustaining activation of PI3K at the post-entry stage was important for virus replication. Though the virus titer reduced 0.6 log compared to the control when it was inhibited at the virus entry stage (Figure 5b), we inferred that the inhibition of PI3K had minor effect on the virus entry. Since the PI3K inhibitor Ly294002 is a cell permeable compound, when it was used in the entry stage, the intracellular residues had some effect on the subsequent stage of the virus replication. Studies currently in progress would show if this was indeed the case. Thus these data suggest that PI3K activation played an important role in fully efficient replication, especially at the post-entry stage. This study provides new insight into our understanding of the interplay between virus BoHV-1 and MDBK cells signaling pathways induced upon infection.
Recent work indicated that many virus exploit host cell signaling pathways to facilitate various steps of virus infection. The PI3K/Akt pathway was proved to be critical in regulating vesicular trafficking for Ebola virus at the entry step. In addition, blocking phosphorylation of Akt at the early step nearly aborted virus replication . Influenza A virus triggered PI3K/Akt pathway activation only at the late phase in infection of human lung carcinoma cells (A549), and the activation has been shown to be required for efficient virus replication . This report indicates that BoHV-1 infection of MDBK cells led to biphasic activation of PI3K/Akt and MAPK/Erk1/2 pathways and Inhibition of PI3K/Akt pathway greatly reduced the virus production (Figure 4a). This evidence was the first to imply that activation of PI3K/Akt is important for BoHV-1 to complete its life cycle. For many viruses, the MAPK/Erk1/2 pathway had been proved to take part in the regulation of gene expression and replication. For example, a bi-phasic Erk1/2 is activated to facilitate Coxsackievirus B3 and human cytomegalovirus infection [19, 15]. Treatment of the cells with MAPK stimulators, such as serum and phorbol myristate acetate to activate MAPK/Erk1/2 signaling pathway could enhanced the replication and infectivity of HIV-1 and BK polyomavirus virus [16, 31]. However, inhibition of MAPK/Erk1/2 pathway had a minor effect on BoHV-1 replication, though biphasic activation was also induced by the virus (Figure 4b and Figure 2a). To explain this, we speculated that during the process of the virus infection once the signaling pathway was inhibited simultaneously or subsequently, another signaling pathway(s) was (were) activated which could replenish the function of MAPK/Erk1/2 pathway. However, further research on this matter is needed.
Shortly after exposure of cells to UV-irradiated virus, which was capable of receptor binding and endocytosis, the early PI3K/Akt and MAPK/Erk1/2 pathways activation was induced, but it was insufficient to induce the late stage phosphorylation (Figure 3). The mechanism for the rapid activation of the two pathways by BoHV-1 was unknown. These activations may be induced by direct virus and cellular receptor(s) and/or co-receptor(s) interaction, such as that seen in the HIV-1 activation of Erk , or by exposure to a viral protein such as the HIV-1 Tat protein . Furthermore, phosphorylation of Akt and Erk1/2 with high level was observed as early as 5 min postinfection strongly indicating that the virus could actively induce the signaling pathways and virus replication was not required for these inductions. Inversely, UV-irradiated virus, incapable of replication, could not active the late-phase activation of the two pathways. Obviously the viral gene expression was responsible for this event as reported in influenza A virus and human cytomegalovirus infection [6, 13].
In summary, our investigation indicates that PI3K/Akt and MAPK/Erk1/2 signaling pathways are induced as a consequence of BoHV-1 infection of MDBK cells. In particular, the activation of PI3K was required for fully efficient replication, especially at the late step. It enhanced our knowledge on the mechanism of the virus infection. The complex interaction between the virus and cellular components during the course of BoHV-1 infection needs to be more clearly defined in the future by focusing on the signaling pathway.