Vaccinia Virus‐Induced Microtubule‐Dependent Cellular Rearrangements

Abstract

Although infection with vaccinia virus (VV) is known to affect the cytoskeleton, it is not known how this affects the cellular architecture or whether the attenuated modified VV ankara (MVA) behaves similar to wild-type VV (wtVV). In the present study, we therefore compared effects of wtVV and MVA infection on the cellular architecture. WtVV-infection induces cell rounding early in infection, which coincides with the retraction of microtubules (MTs) and intermediate filaments from the cellular periphery, whereas mitochondria and late endosomes cluster around the nucleus. Nocodazole treatment demonstrates that cell rounding and organelle clustering require intact MTs. At the onset of virus assembly late in infection, cells reflatten, a process that coincides with the regrowth of MTs into the cellular periphery. We find that the actin network undergoes several rearrangements that occur sequentially in time and that closely follow the cell-shape changes. Unexpectedly, these actin changes are blocked or reversed upon nocodazole treatment, indicating that intact MTs are also responsible for the wtVV-induced actin rearrangements. Finally, MVA infection does not induce any of these cellular changes. Because this virus lacks a substantial number of VV genes, MVA opens up a system to search for the molecules involved in wtVV-induced cellular changes; in particular, those that may regulate actin/MT interactions.Key words: actin, cell shape, microtubules, modified vaccinia virus ankara, vaccinia virus. Vaccinia virus (VV) is the prototype of the poxviridae, a family of large DNA viruses. Its genome of about 200 kB encodes for more than 250 proteins (1) enabling VV to carry out DNA replication and transcription in the cytoplasm rather than in the cellular nucleus (1). VV is characterized by a complex cytoplasmic life cycle, directed by three classes of genes, early, intermediate and late. Following entry, the viral core is delivered into the cytoplasm, from which a defined set of early mRNAs is transcribed and extruded into the cytoplasm for translation. The early proteins are required to uncoat the viral core and to initiate cytoplasmic DNA replication. Replication sets off the transcription of intermediate and late genes, late proteins being required for virion assembly. During assembly, late in infection, two infectious forms are made; the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). A small percentage of the intracellular IMVs becomes enwrapped by a double membrane derived from the transGolgi network (TGN) or endosomes to form the intracellular enveloped virus [IEV (2,3)]. The IEV moves along microtubules (MTs) towards the plasma membrane using the plusend directed motor protein kinesin-1. Upon fusion of the outer IEV membrane with the plasma membrane, the EEV is released into the extracellular environment. The outer IEV membrane that is fused with the plasma membrane is able to polymerize actin tails, resulting in long filopodia that propel attached EEVs towards neighbouring cells, a pr...

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

“…4a to l) and are in agreement with data reported by others (21,27,28). These virus-induced alterations, however, were less pronounced in the JNK1/2-KO cells (Fig.…”

“…4f and e, insets). These data are consistent with previous observations (21,26,27,28), and morphotype 1 predominated at between 3 and 6 hpi (Fig. 4, graph 1) and at those times represented about 70 and 55% of the cells, respectively.…”

“…In agreement with the viral need for early gene expression to stimulate cell migration (26,27,28), we provided evidence that this event was significantly affected in the MKK/JNK-KO cells (Fig. 5B and C).…”

“…During the VACV infectious cycle, infected cells undergo migration, a process that not only is dependent on the virus-cell ligand interaction but that also requires expression of viral early genes (26,27,28) and repression of RhoA signaling by the viral protein F11L (34). During our analysis of VACV-induced cytopathic effects, typical migrating cells were observed in both WT and KO cells.…”

“…It has been known that during the course of VACV infection both MT and actin networks undergo accentuated modifications that facilitate the inward and outward movement of viral particles (21,24,26,27,28,38). To investigate whether JNK is associated with cytoskeleton alterations that follow VACV infection, WT and JNK1/2-KO cells were either uninfected or infected with VACV (MOI, 5).…”