Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

Abstract

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

“…We did not observe complete inhibition of nuclear entry at this time point, suggesting either reversible inhibition of nuclear import by WGA, which has previously been reported (73), or an alternative, NPC-independent pathway that is utilized by rAAV2. The classical import pathway described herein for rAAV2 differs from the mechanism of nuclear entry proposed by Cohen et al for a related parvovirus, MVM (45), and also recent findings describing nuclear envelope breakdown of permeabilized HeLa cells by AAV2 that had been acidified and then neutralized (50). Future work is required to understand how the acidification and neutralization of rAAV2 mediates nuclear entry under physiological subcellular trafficking conditions.…”

“…Recently, a PDZbinding domain was discovered on the N terminal of VP1 that was shown to be important for nuclear entry of wt AAV2 (49). While the current study was under review, a recent study revealed that AAV2 that had been acidified and then neutralized could cause nuclear envelope breakdown in permeabilized HeLa cells (50). As in vitro assays become more harmonized with respect to physiological infection, these results, taken together, suggest that AAV may utilize multiple import mechanisms to gain access to the nucleus, or that the mechanism of nuclear entry might vary for wt AAV2 and AAV vectors.…”

“…Moreover, previous work has shown that AAV2 can interact with the nucleolar proteins nucleophosmin and nucleolin (27,90,91), suggesting that rAAV2 can translocate into the nucleus through interaction with these proteins. In addition to the observations of nuclear envelope breakdown by parvoviruses, including AAV2, Porwal et al showed that AAV2 can directly interact with nucleoporins that comprise the nuclear pore complex (50). Finally, given that the abundance of karyopherins differs among cell types, it is possible that rAAV serotypes could utilize different karyopherins for nuclear entry, which would further contribute to the differences in cellular tropism historically observed among the rAAV serotypes.…”

Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

“…To induce perforation of the nuclear membranes without mitosis, we made use of parvovirus H1, a small DNA virus known to enter the nucleus by inducing holes in the NE [64]. These hole are large enough to allow sudden entry of antibodies into the nucleus and, at least in some cells, chromatin leakage into the cytoplasm.…”

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

“…In early steps, incoming capsids need to either enter the nucleus or at least dock to the nuclear membrane to inject the viral genome into the nucleus (18)(19)(20)(21). In late steps of infection, newly synthesized capsid proteins rapidly assemble into trimers before nuclear transport (22,23).…”