Many viruses have been reported to exploit the host cellular machinery throughout their life cycle due to their parasitic nature and simplicity . Several reports showed that the cytoskeleton plays an important role in the intracellular traffic of some viruses [9, 23–26]. Frog virus 3 (FV3) was found to interact with the cytoskeleton and disrupt the actin cytoskeleton at the initial stages of infection . Treatment of infected cells with cytochalasin has been demonstrated to affect the release of FV3 at the plasma membrane level. Tiger frog virus (TFV) was reported to cause the reorganization of microtubules in infected zebrafish embryo fibroblast 4 (ZF4) cells .
In the present study, we found that depolymerization of the actin filaments with cyto D, cyto B, or lat A reduced ISKNV infection, the virus blockage at the entry step of its life cycle potentially caused the reduced ISKNV infection. In addition, the depolymerization of actin filaments reduced both the total amount of virus produced in the cell and the amount of virus that was allowed to egress from cells in the late stages of ISKNV infection. These data demonstrate that ISKNV relies on an intact actin network during infection.
Increasing evidence has showed that the actin cytoskeleton is involved in many endocytic pathways, although to varying degrees . Entry by endocytosis may require remodeling of the actin cytoskeleton, while fusion at the cell surface might not rely as heavily on the actin cytoskeleton . Our results showed that microfilament depolymerization did not change virus binding to the cell, but it efficiently inhibited virus internalization. Many previous reports have demonstrated that microfilaments are dispensable for viral binding to the host cell [30–32]. The role of microfilaments in viral internalization may be useful to better understand the precise entry mechanism of ISKNV.
Actin filaments have been shown to be essential for infection by several other viruses [23, 33, 34]. Using inhibitor depolymerizing actin filaments, we evaluated the effect of disrupting actin systems on the infectivity of ISKNV. Our results indicated that disruption of microfilaments with cyto D, cyto B, or lat A inhibited the infection of MFF-1 cells by ISKNV. Furthermore, using qPCR, we found that disrupting microfilaments inhibited early steps of virus entry. However, the disruption of microfilaments could not inhibit the virus entry completely, which could be attributed to a caveola-mediated internalization mechanism through which ISKNV enters MFF-1 cells. Similar to other viruses, ISKNV might use more than one route to enter cells. In this case, inhibition of one pathway might not affect viral entry via another pathway, resulting in a reduced number of viral particles entering the cells . In fact, cells have been demonstrated to upregulate alternate endocytic routes if an endocytic pathway is blocked . Moreover, caveolae and caveolin-associated signaling proteins and receptors have been reported to be linked to a dynamic filamentous actin network via structural proteins . The disruption of actin may destroy the caveola-mediated internalization mechanism through which ISKNV enters MFF-1 cells and then impede ISKNV infection. Further studies are needed to clarify the role of actin in caveola-mediated endocytosis during ISKNV entry and trafficking in MFF-1 cells.
We also sought to determine the effect of inhibitors on later stages of viral replication. In the present study, we evaluated the replication ability of ISKNV in presence of actin inhibitors and found a significant reduction in virus replication. These results indicate that the microfilaments are possibly involved in an interaction with the viral replication machinery. Several reports have shown that actin microfilaments participate in late stages of viral replication, such as assembly and release . Treatment with the cyto D, the Autographa californica nucleopolyhedrovirus budding from host cells was drastically inhibited . Cyto D caused numerous microvillus-like projections containing virions and actin microfilaments to accumulate on the infected cell surface in the late stage of frog virus 3 infections . The utilization of a cellular cytoarchitecture for viral replication has also been reported in several viruses, such as human parainfluenza virus type 3 , mouse mammary tumor virus , and measles virus . To date, little is known about the accurate kinetics of ISKNV replication cycle. Our results showed that treatment with cyto D and cyto B reduced total ISKNV production (Figure 4), but which late step(s) of the viral life was affected by microfilaments should be further studies. All these results suggested that actin filaments played an important role in viral replication cycle in vitro using the MFF-1 cell line.
In addition, many viruses may employ the actin and microtubule network to transport their nucleocapsids protein . Nucleocapsids of the murine mammary tumor virus have been found to interact with actin with this interaction reported to be necessary for extruding virus particles from infected cells . Xiong et al. (2011) suggested that the ISKNV major capsid protein (MCP) gene interacts with the β-actin of zebrafish. In our study, we also find that the actin of MFF-1 cells interacts with the MCP of ISKNV by co-immunoprecipitation (data not shown). All the results provide strong evidence that the actin network potentially participates in ISKNV intracellular traffic and the release of virus from cells.