ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.

The multicomponent type VI secretion system (T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/VipB tubules are decorated by ClpV in vivo and become static in ΔclpV cells, indicating that ClpV is required for tubule removal. VipA/VipB tubules mislocalize in ΔclpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/VipB tubules. ClpV activity is restricted to the contracted state of VipA/VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/VipB conformation.

Vaccinia-virus-induced cellular contractility facilitates the subcellular localization of the viral replication sites.

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-motor-mediated 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.

Vaccinia virus-induced microtubule-dependent cellular rearrangements.

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.

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum.

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.