Vaccinia‐Virus‐Induced Cellular Contractility Facilitates the Subcellular Localization of the Viral Replication Sites

Abstract

Poxviruses, such as vaccinia virus (VV), replicate their DNA in endoplasmic-reticulum-enclosed cytoplasmic sites. Here, we compare the dynamics of the VV replication sites with those of the attenuated strain, modified VV Ankara (MVA). By live-cell imaging, small, early replication sites of both viruses undergo motility typical of microtubule (MT)-motor-mediated movement. Over time, growing replication sites of VV collect around the nucleus in a MT-dependent fashion, whereas those of MVA remain mostly scattered in the cytoplasm. Surprisingly, blocking the dynein function does not impair the perinuclear accumulation of large VV replication sites. Live-cell imaging demonstrates that in contrast to small replication sites, large sites do not display MT-motormediated motility. Instead, VV infection induces cellular contractility that facilitates the collection of growing replication sites around the nucleus. In a subset of cells (30-40%), this VV-induced contractility is alternated by phases of directed cell migration, suggesting that the two processes may be linked. The MVA-infected cells do not display contractility or cell migration, supporting the idea that these cellular activities facilitate the efficient accumulation of the VV replication sites around the nucleus. We propose that the recently described cytoskeletal rearrangements induced by VV are a prerequisite for the observed cell contractility and migration activities that apparently contribute to the organization of the complex cytoplasmic life cycle of VV.

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

“…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.…”

“…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).…”

Vaccinia Virus Particles Mix Inefficiently, and in a Way That Would Restrict Viral Recombination, in Coinfected Cells

“…Confocal microscopy has been used to detect virosomal substructures, specifically DNA-free channels, and the pattern of proteins expressed from each virosome suggests that most are composed of DNA encompassing a single virus genotype (17). This is consistent with the observation that few mixing events are seen in cells bearing more than one virosome and followed using static (17) or live cell imaging (27). In this communication, we examine in greater detail the extent of interaction between virosomes in coinfected cells and show that the intracellular milieu creates constraints that limit the fusion of coinfecting virus particles and the mixing of different viral DNAs.…”

“…A particular problem is that VAC-infected cells round up and move in an unpredictable manner during infection (27), and if some z planes are not captured in all of the scans over all of the time points, then some smaller virosomes could be missed and their disappearance possibly recorded as fusion. Thus, one cannot reliably track all of the fusion events as a function of virosome number.…”