It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.

Summary Adult stem cells reactivate from quiescence to maintain tissue homeostasis and in response to injury. How the underlying regulatory signals are integrated is largely unknown. Drosophila neural stem cells (NSCs) also leave quiescence to generate adult neurons and glia, a process that is dependent on Hippo signaling inhibition and activation of the insulin-like receptor (InR)/PI3K/Akt cascade. We performed a transcriptome analysis of individual quiescent and reactivating NSCs harvested directly from Drosophila brains and identified the conserved STRIPAK complex members mob4 , cka , and PP2A ( microtubule star , mts ). We show that PP2A/Mts phosphatase, with its regulatory subunit Widerborst, maintains NSC quiescence, preventing premature activation of InR/PI3K/Akt signaling. Conversely, an increase in Mob4 and Cka levels promotes NSC reactivation. Mob4 and Cka are essential to recruit PP2A/Mts into a complex with Hippo kinase, resulting in Hippo pathway inhibition. We propose that Mob4/Cka/Mts functions as an intrinsic molecular switch coordinating Hippo and InR/PI3K/Akt pathways and enabling NSC reactivation.

We report the efficacy of rapamycin by reducing CSCs proliferation and tumorigenic potential in vitro. Despite these encouraging results, the efficacy in vivo was very poor. This finding confirms the limited use of rapamycin as a monotherapy for glioblastomas.